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The inhibition of aldosterone activity is a useful approach for preventing the progression of cardiovascular and renal diseases in hypertensive patients. Although the results of our previous in vivo study suggested that N-type calcium channels may have a role in regulating plasma aldosterone levels, the direct relationship between N-type calcium channels and aldosterone production in adrenocortical cells has not been examined. In this study, the analysis of quantitative reverse transcription-PCR, western blotting, and immunocytological staining indicated the possible presence of N-type calcium channels in human adrenocortical cells (H295R cell line). Patch clamp analysis indicated that omega-conotoxin GVIA (CnTX), an N-type calcium channel inhibitor, suppressed voltage-dependent barium currents. During steroidogenesis, CnTX significantly reduced the transient calcium signaling induced by angiotensin II (Ang II) and partially prevented Ang II-induced aldosterone and cortisol formation with no significant influence on CYP11B2 and CYP11B1 mRNA expression. In addition, in α1B calcium channel subunits, knockdown significantly decreased Ang II-induced aldosterone formation with increments in CYP11B2 mRNA expression. We also investigated the inhibitory activities of some types of dihydropyridine calcium channel blockers (CCBs; cilnidipine: L-/N-type CCB, efonidipine: L-/T-type CCB, and nifedipine: L-type CCB), and these agents showed a dose-dependent inhibition effect on Ang II-induced aldosterone and cortisol production. Furthermore, only cilnidipine failed to suppress CYP11B1 expression in H295R cells. These results suggest that N-type calcium channels have a significant role in transducing the Ang II signal for aldosterone (and cortisol) biosynthesis, which may explain the mechanism by which N-type calcium channels regulate plasma aldosterone levels.

Background: Our previous study suggests that decreased P-450(c17alpha) expression correlated with the overproduction of aldosterone in APA and nodular hyperplasia in patients with primary aldosteronism. This study was performed to further investigate if P-450(c11beta) contributes to the overproduction of aldosterone in APA and nodular hyperplasia tissues., Methods: Total RNA and protein were extracted from 7 cases of APA tissue, 3 nodular hyperplasia tissues, 7 normal adrenal glands. P-450(c11beta) mRNA was examined by dot blot and confirmed by Northern blot analysis and by realtime PCR. Protein expression level of P-450(c11beta) was also investigated by immunohistochemical staining and confirmed by Western blot., Results: The relative expression level of P-450(c11beta) mRNA to beta-actin in APA, nodular hyperplasia and the normal adrenal gland group are 47 +/- 22%, 55 +/- 13%, 64 +/- 16% respectively by dot blot and are 94 +/- 18%, 101 +/- 20%, 112 +/- 62% respectively by Northern blot. These results are further confirmed by realtime PCR. This result was also supported by the relative protein expression level of P-450(c11beta) to beta-actin which are 118 +/- 15%, 107 +/- 32%, 108 +/- 22% respectively evaluated by Western blot. There was no significant difference in protein expression level of P-450(c11beta) among the normal adrenal gland tissues, APA and adrenal nodular hyperplasia tissue, either (P > 0.05)., Conclusions: These results suggest that P-450(c11beta) is not a key contributor to the overproduction of aldosterone in APA and nodular hyperplasia and can not be considered as a potential marker to differentiate between them in patients with primary aldosteronism.

A series of 23 N-(Pyridin-3-yl)benzamides was synthesized and evaluated for their potential to inhibit human steroid-11β-hydroxylase (CYP11B1) and human aldosterone synthase (CYP11B2). The most potent and selective CYP11B2 inhibitors (IC(50) values 53-166 nM) were further evaluated for their potential to inhibit human CYP17 and CYP19, and no inhibition was observed. Clear evidence was shown for N-(Pyridin-3-yl)benzamides to be a highly selective class of CYP11B2 inhibitors in vitro., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Aldosterone biosynthesis is not only altered in rare mendelian disorders. Recent evidence suggests that common polymorphisms in the genes mediating the final stages of aldosterone and cortisol production (CYP11B1 and CYP11B2 respectively) are also associated with milder alterations in adrenal corticosteroid biosynthesis. These abnormalities consist of a decrease in adrenal 11β- hydroxylase activity and a subtle, life-long excess of aldosterone secretion which may lead to long-term cardiovascular risks. An interaction between the CYP11B1 and CYP11B2 genes may exist but is yet to be elucidated. This article describes the studies which highlight the importance of adrenal steroid synthesis in the development of hypertension and cardiovascular dysfunction as well as the role of common polymorphisms in adrenal synthetic genes in altering corticosteroid biosynthesis., (Copyright © 2011 S. Karger AG, Basel.)

To understand the mechanism of sex differentiation in the protogynous Malabar grouper Epinephelus malabaricus, we performed an immunohistochemical investigation of the expression of three steroidogenic enzymes, cholesterol-side-chain-cleavage enzyme (CYP11a), aromatase (CYP19a1a), and cytochrome P45011beta-hydroxylase (CYP11b), in the gonads during ovarian differentiation. Strong positive immunoreactivity against CYP11a, the key enzyme of steroidogenesis, and CYP19a1a which is essential for estrogen (17beta-estradiol) production, appeared first in the somatic cells surrounding gonial germ cells in undifferentiated gonads and throughout ovarian differentiation. However, positive immunoreactivity against CYP11b, which is important for androgen (11-ketotestosterone) production, first appeared in the cluster of somatic cells in the ovary tunica near the dorsal blood vessel after differentiation. CYP19a1a and CYP11b did not co-localize in any cells. These results indicate that there are two types of steroid-producing cells, estrogen-producing cells and androgen-producing cells, in the gonads of this fish, and they are distributed differently, suggesting that these cells are derived from different somatic cells. Estrogen-producing cells appeared prior to ovarian differentiation, while androgen-producing cells were first detected after ovarian differentiation. These results suggest that endogenous estrogen is involved in ovarian differentiation.

The orphan nuclear receptor SF-1 (steroidogenic factor 1) is highly expressed in the pituitary, gonad and adrenal glands and plays key roles at all levels of the hypothalamic-pituitary-steroidogenic tissue axis. In the present study, we show that PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator 1α] interacts with and co-activates SF-1 to induce LHβ (luteinizing hormone β) and αGSU (α-glycoprotein subunit) gene expression, subsequently leading to the increased secretion of LH in pituitary gonadotrope-derived αT3-1 cells. PGC-1α co-activation of LHβ expression requires an SF-1-binding element [GSE (gonadotrope-specific element)] mapped to the promoter region of LHβ. Mammalian two-hybrid and co-immunoprecipitation assays, as well as GST (glutathione transferase) pull-down experiments demonstrated that PGC-1α interacts with SF-1 in vivo and in vitro. Additionally, PGC-1α stimulates the expression of Cyp11b2 (aldosterone synthase gene), Cyp11b1 (steroid 11β-hydroxylase gene) and P450scc (cholesterol side-chain cleavage enzyme), and the synthesis of aldosterone in adrenal-cortex-derived Y-1 cells. Chromatin immunoprecipitation assays confirmed that endogenous PGC-1α co-localizes with SF-1 in the LHβ and Cyp11b2 promoter region. Knockdown of endogenous SF-1 by siRNA (small interfering RNA) abolished the PGC-1α induction of LHβ and Cyp11b2 gene expression in αT3-1 and Y-1 cells respectively. Finally, we demonstrated that PGC-1α induces SF-1 gene expression in both αT3-1 and Y-1 cells. Taken together, our findings reveal the potential role of PGC-1α and suggest that it may play important roles in steroidogenesis, gonad development and sex differentiation through SF-1.

Alkylphenol ethoxylate, consisting of ∼80% nonylphenol ethoxylate (NPEO), is a major group of nonionic surfactant. The primary degradation product of NPEO, nonylphenol (NP), interferes with reproduction, induces cell death in gonads, and leads to changes in other reproductive parameters. With such apparent stress, NP is believed to induce stress response mechanism, i.e., adrenal cortical hormone. However, the effects and action mechanisms of NP on rat adrenal zona fasciculata-reticularis (ZFR) cells are still unclear. This study explored the effects of NP on corticosterone release. ZFR cells were incubated with NP in the presence or absence of adrenocorticotropin (ACTH), 8-bromo-cyclic 3',5'-adenosine monophosphate (8-Br-cAMP), forskolin (FSK), 25-hydroxyl cholesterol (25-OH-cholesterol), pregnenolone, progesterone, or deoxycorticosterone at 37°C for 1 h. The concentrations of corticosterone or pregnenolone in the spent media were measured by radioimmunoassay. The expressions of steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage (P450scc) protein, and 11β-hydroxylase in the cells were measured by Western blot. The data demonstrated that (1) NP stimulated corticosterone release induced by ACTH, 8-Br-cAMP, FSK, 25-OH-cholesterol, pregnenolone, progesterone, or deoxycorticosterone; (2) NP significantly increased pregnenolone release in the control, 25-OH-cholesterol, trilostane, and 25-OH-cholesterol + trilostane groups; (3) NP-stimulated corticosterone release was estrogen receptor dependent, but mediated by nitric oxide and p38 mitogen-activated protein kinase pathway independent; and (4) NP did not affect StAR, 11β-hydroxylase, or P450scc protein expression. These results suggest that NP acts directly on rat ZFR cells to stimulate corticosterone release and that the stimulation mechanism of NP mediates through post-cAMP corticosterone manufacture enzymes, i.e., P450scc and 11β-hydroxylase.

Adrenal rest tumor presenting as palpable testicular mass has been well described in boys and adult males with congenital adrenal hyperplasia. It develops most commonly in patients with 21- hydroxylase deficiency, but the entity may also occur in rare forms of congenital adrenal hyperplasia, including 11β-hydroxylase deficiency. Because the management of testicular adrenal rest tumors is substantially different from that applied in benign and malignant testicular tumors, an accurate differentiation between these entities is particularly important. Authors present the history of a young adult male with 11β-hydroxylase deficiency who developed adrenal rest tumors presenting as palpable bilateral testicular masses during treatment with glucocorticoids, then testicular masses showed a rapid regression after an adequate glucocorticoid treatment. Considering lessons obtained from this case, authors review the pathomechanism, symptoms, as well as current diagnostic and treatment modalities of testicular adrenal rest tumors.

Primary aldosteronism is the most common form of secondary hypertension with hypokalemia and suppressed renin-angiotensin system caused by autonomous aldosterone production. Our aim was to compare zona glomerulosa (ZG) structure and function between control adrenals and the peritumoral tissue from patients operated on for aldosterone-producing adenoma. ZG morphology and CYP11B1, CYP11B2, and disabled 2 expression were studied in 15 control adrenals and 25 adrenals with aldosterone-producing adenoma. A transcriptome analysis was done using publicly available data sets. In control adrenals, ZG was discontinuous, and CYP11B2 expression was focal or partly continuous and localized to 3 structures, foci, megafoci, and aldosterone-producing cell clusters. CYP11B2 expression was restricted to a limited number of ZG cells expressing Dab2 but not CYP11B1; aldosterone-producing cell clusters were composed of cells with an intermediate phenotype expressing CYP11B2 but not disabled 2 or CYP11B1. In peritumoral tissue, large remodeling of the adrenal cortex was observed with increased nodulation and decreased vascularization that were not correlated with CYP11B2 expression. In 17 out of 25 adrenals, hyperplasia of adjacent ZG was observed with persistent expression of CYP11B2 that was extended to the entire ZG. In all of the adrenals from patients with aldosterone-producing adenoma, CYP11B2 expression was present in foci, megafoci, and aldosterone-producing cell clusters. Transcriptome profiling indicates a close relationship between peritumoral and control adrenal cortex. In conclusion, adrenal cortex remodeling, reduced vascularization, and ZG hyperplasia are major features of adrenals with aldosterone-producing adenoma. Transcriptional phenotyping is not in favor of this being an intermediate step toward the formation of aldosterone-producing adenoma.

Cholesteryl ester transfer protein (CETP) plays an important role in reverse cholesterol transport and the maintenance of cholesterol homeostasis. The consequences of CETP activity are influenced by triglyceride (TG) levels. When TG levels are increased, CETP promotes transfer of cholesteryl esters to VLDL and the generation of atherogenic dyslipidemia. Combined activities of CETP and hepatic lipase in the presence of TG-rich lipoproteins generate small, dense HDL and LDL particles. Inhibition of CETP may reduce the risk of atherosclerosis in patients with dyslipidemia. Decreasing CETP activity has consistently inhibited atherosclerosis in animal models. Three small-molecule CETP inhibitors, dalcetrapib, torcetrapib, and anacetrapib, have been or are being tested in phase 3 clinical studies. All three demonstrated potentially beneficial effects on the lipid profile in patients with dyslipidemia. Imaging studies with torcetrapib failed to show an effect on atherosclerosis in humans, probably because of the off-target effect on the RAAS. Preclinical evidence suggests that dalcetrapib seems to lack the off-target adverse effects on the RAAS that potentially caused the clinical development of torcetrapib to be halted. The lack of adverse effects on the RAAS remains to be confirmed in phase 3 studies with dalcetrapib. CETP inhibition as a therapeutic strategy remains a valid option to be tested with a CETP inhibitor that does not share torcetrapib's off-target effects., (Copyright © 2010 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

The current study presents data indicating that 1alpha,25-dihydroxyvitamin D(3) affects the production of hormones and expression of crucial steroidogenic enzymes in the human adrenocortical cell line NCI-H295R. This cell line is widely used as a model for adrenal steroidogenesis. Treatment of the cells with 1alpha,25-dihydroxyvitamin D(3) suppressed the levels of corticosterone, aldosterone, DHEA, DHEA-sulfate and androstenedione in the culture medium. In order to study the mechanisms behind this suppression of hormone production, we investigated the effects of 1alpha,25-dihydroxyvitamin D(3) on important genes and enzymes controlling the biosynthesis of adrenal hormones. The mRNA levels were decreased for CYP21A2 while they were increased for CYP11A1 and CYP17A1. No significant changes were observed in mRNA for CYP11B1, CYP11B2 or 3beta-hydroxysteroid dehydrogenase (3betaHSD). In similarity with the effects on mRNA levels, also the endogenous enzyme activity of CYP21A2 decreased after treatment with 1alpha,25-dihydroxyvitamin D(3). Interestingly, the two CYP17A1-mediated activities were influenced reciprocally - the 17alpha-hydroxylase activity increased whereas the 17,20-lyase activity decreased. The current data indicate that the 1alpha,25-dihydroxyvitamin D(3)-mediated decrease in corticosterone and androgen production is due to suppression of the 21-hydroxylase activity by CYP21A2 and the 17,20-lyase activity by CYP17A1, respectively. In conclusion, the current study reports novel findings on 1alpha,25-dihydroxyvitamin D(3)-mediated effects on hormone production and regulation of genes and enzymes involved in steroidogenesis in the adrenocortical NCI-H295R cell line, a model for human adrenal cortex., (2010 Elsevier B.V. All rights reserved.)

Aldosterone, the final component of the renin-angiotensin-aldosterone system, plays an important role in the pathophysiology of hypertension and congestive heart failure. Aldosterone synthase (CYP11B2) catalyzes the last three steps of aldosterone biosynthesis, and as such appears to be a target for the treatment of these disorders. A sulfonamide-imidazole scaffold has proven to be a potent inhibitor of CYP11B2. Furthermore, this scaffold can achieve high levels of selectivity for CYP11B2 over CYP11B1, a key enzyme in the biosynthesis of cortisol., (Copyright 2010 Elsevier Ltd. All rights reserved.)

To characterize intraadrenal adaptations for inhibition of cortisol synthesis, we analyzed the effects of etomidate (ETO) on steroid hormone secretion and expression of key regulators of steroidogenesis and proliferation in human NCI-h295 adrenocortical cancer cells. Etomidate potently blocked 11beta-hydroxylase (CYP11B1), aldosterone synthase (CYP11B2), and side chain cleavage enzyme (CYP11A1). This inhibition of steroidogenesis was associated with increased expression of steroidogenic acute regulatory protein (StAR), and CYP11A1 and 17alpha-hydroxylase/17, 20-lyase (CYP17A1) protein levels, but not of the respective mRNA levels. Promoter activity of CYP11A1 and melanocortin 2 receptor (MC2R) was not increased by etomidate in treated cells compared to controls. The increase in protein levels was partially reversed by cycloheximide suggesting post-transcriptional mechanisms but also protein stabilization as underlying cause. Furthermore, ETO exhibited antiproliferative activity paralleled by a decrease in phosphorylation of MEK and ERK1, 2. In summary, ETO exhibits pleiotropic effects on adrenal function in vitro. Inhibition of steroidogenesis is followed by increased levels of steroidogenic key proteins and reduced proliferation. These changes reflect adaptations to maintain steroidogenesis at the cost of adrenal proliferation., (Georg Thieme Verlag KG Stuttgart * New York.)

A data set of a series of 132 structurally diverse compounds with cytochrome 11B2 and 11B1 (CYP11B2 and CYP11B1) enzyme inhibitory activities was subjected to molecular shape analysis to explore contributions of shape features as well as electronic, structural, and physicochemical parameters toward enzyme inhibitory activities, in search of appropriate molecular scaffolds with optimum substitutions for highly potent CYP11B2 inhibitors. Genetic function approximation (GFA) and genetic partial least squares (G/PLS) were used as chemometric tools for modeling, and the derived equations were of acceptable statistical quality considering both internal and external validation parameters (Q(2): 0.514-0.659, R(2)(pred): 0.510-0.734). The G/PLS models with spline option for CYP11B2 and CYP11B1 inhibition and selectivity modeling appeared to be the best models based on r(m)(2)((overall)) criterion. The study indicates the importance of the pyridinylnaphthalene and pyridylmethylene-indane scaffolds with less polar and electrophilic substituents for optimum CYP11B2 inhibitory activity and CYP11B2/CYP11B1 selectivity.

The vocalizing midshipman fish, Porichthys notatus, has two male morphs that exhibit alternative mating tactics. Only territorial males acoustically court females with long duration (minutes to >1h) calls, whereas sneaker males attempt to steal fertilizations. During the breeding season, morph-specific tactics are paralleled by a divergence in relative testis and vocal muscle size, plasma levels of the androgen 11-ketotestosterone (11KT) and the glucocorticoid cortisol, and mRNA expression levels in the central nervous system (CNS) of the steroid-synthesizing enzyme aromatase (estrogen synthase). Here, we tested the hypothesis that the midshipman's two male morphs would further differ in the CNS, as well as in the testis and vocal muscle, in mRNA abundance for the enzymes 11beta-hydroxylase (11betaH) and 11beta-hydroxysteroid dehydrogenase (11betaHSD) that directly regulate both 11KT and cortisol synthesis. Quantitative real-time PCR demonstrated male morph-specific profiles for both enzymes. Territorial males had higher 11betaH and 11betaHSD mRNA levels in testis and vocal muscle. By contrast, sneaker males had the higher CNS expression, especially for 11betaHSD, in the region containing an expansive vocal pacemaker circuit that directly determines the temporal attributes of natural calls. We propose for territorial males that higher enzyme expression in testis underlies its greater plasma 11KT levels, which in vocal muscle provides both gluconeogenic and androgenic support for its long duration calling. We further propose for sneaker males that higher enzyme expression in the vocal CNS contributes to known cortisol-specific effects on its vocal physiology., (Copyright 2010 Elsevier Inc. All rights reserved.)

Context: Aldosterone synthase (CYP11B2) and steroid 11 beta-hydroxylase (CYP11B1) catalyze the terminal steps for aldosterone and cortisol syntheses, respectively, thereby determining the functional differentiation of human adrenocortical cells. Little is known, however, about how the cells expressing the enzymes are actually distributed in the adrenals under normal and pathological conditions., Objective: The objective of the study was to determine the localization of CYP11B2 and -B1 in human adrenal specimens by using developed antibodies capable of distinguishing the two enzymes from each other., Results: Under normal conditions, CYP11B2 was sporadically detected in the zona glomerulosa, whereas CYP11B1 was entirely detected in the zonae fasciculata-reticularis. Adrenocortical cells lacking both enzymes were observed in the outer cortical regions. In addition to conventional zonation, we found a variegated zonation consisting of a subcapsular cell cluster expressing CYP11B2, which we termed aldosterone-producing cell cluster, and a CYP11B1-expressing area. Aldosterone-producing adenomas differed in cell populations expressing CYP11B2 from one another, whereas CYP11B1-expressing and double-negative cells were also intermingled. Adenomas from patients with Cushing's syndrome expressed CYP11B1 entirely but not CYP11B2, resulting in atrophic nontumor glands. The nontumor portions of both types of adenomas bore frequently one or more aldosterone-producing cell clusters, which sustained CYP11B2 expression markedly under the conditions of the suppressed renin-angiotensin system., Conclusion: Immunohistochemistry of the human normal adrenal cortex for CYP11B2 and CYP11B1 revealed a variegated zonation with cell clusters constitutively expressing CYP11B2. This technique may provide a pathological confirmatory diagnosis of adrenocortical adenomas.

Infants rarely present with truly ambiguous genitalia and such children should be evaluated by experts who work within a multidisciplinary team that is dedicated for evaluation and management of children and adults with suspected and confirmed disorders of sex development. The paediatric endocrinologist who is a vital and often the central member of this clinical team not only needs to lead the clinical evaluation of the infant systematically but also needs to be sensitive to the needs of the infant, the parents and the rest of the team. A thorough knowledge of the underlying pathophysiology and the strengths and weaknesses of the investigative tools that are available for reaching a diagnosis is crucial., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

It is hypothesized that blocking the adrenocorticotrophic hormone (ACTH) receptor, using either a blocking antibody or a drug will result in a medical cortical-adrenalectomy, with relative sparing of mineralocorticoid hormone production. This would be similar to the clinical findings in familial glucocorticoid deficiency type 1, an autosomal recessive condition due to inactivating mutations of the adrenal receptor for ACTH, also known as the melanocortin 2 receptor (MC2R). It is further hypothesized that MC2R blockade should allow using lower glucocorticoid doses to treat congenital adrenal hyperplasia (CAH) due to enzyme deficiency of either 21-hydroxylase (CYP21B) or 11-hydroxylase (CYP11B1), thus reaching a better final adult height than with current therapeutic strategies. Blocking the ACTH receptor can also be employed to medically treat Cushing's due to excess pituitary or ectopic ACTH production., ((c) 2009 Elsevier Ltd. All rights reserved.)

Aspergillus tamarii contains an endogenous lactonization pathway which can transform progesterone to testololactone in high yield through a sequential four step enzymatic pathway. In this pathway testosterone is formed which primarily undergoes oxidation of the C-17beta-alcohol to a C-17 ketone but, can also enter a minor hydroxylation pathway where 11beta-hydroxytestosterone is produced. It was recently demonstrated that this hydroxylase could monohydroxylate 3beta-hydroxy substituted saturated steroidal lactones in all four possible binding orientations (normal, reverse, inverted normal, inverted reverse) on rings B and C of the steroid nucleus. It was therefore of interest to determine the fate of a series of 3alpha-substituted steroidal analogues to determine stereochemical effect on transformation. Hydroxylation on the central rings was found to be restricted to the 11beta-position (normal binding), indicating that the 3alpha-stereochemistry removes freedom of binding orientation within the hydroxylase. The only other hydroxylation observed was at the 1beta-position. Interestingly the presence of this functional group did not prevent lactonization of the C-17 ketone. In contrast the presence of the 11beta-hydroxyl completely inhibited Baeyer-Villiger oxidation, a result which again demonstrates that single functional groups can exert significant control over metabolic handling of steroids in this organism. This may also explain why lactonization of 11beta-hydroxytestosterone does not occur. Lactonization of the C-17 ketone was not significantly affected by the 3alpha-alcohol with significant yields achieved (53%). Interestingly a time course experiment demonstrated that the presence of the 3alpha-acetate inhibited the Baeyer-Villiger monooxygenase with its activity being observed 24h later than non-acetate containing analogues. Apart from oxidative transformations observed a minor reductive pathway was revealed with the C-17 ketone being reduced to a C-17beta-alcohol for the first time in this organism.

Oleoyl-estrone (OE) induces a marked loss of body fat in rats by maintaining energy expenditure, body protein and blood glucose despite decreasing food intake. OE increases glucocorticoids, but they arrest OE lipid-mobilization. We studied here whether OE induces a direct effect on adrenal glands function as part of this feedback regulation. Dietary overweight male rats were given oral 10nmol/g OE gavages for ten days. A group (PF) of pair-fed to OE rats, and controls received vehicle-only gavages. OE rats lost slightly more body than PF, but had larger adrenal glands. Tissue corticosterone levels, and gene expressions for glucocorticoid-synthesizing enzymes were increased in OE versus controls and PF; thus, we assumed that adrenal growth affected essentially its cortex since OE also lowered the expression of the medullar catecholamine synthesis enzyme genes. Serum corticosterone was higher in PF than in OE and controls, but liver expression of corticosteroid-disposing steroid 5alpha-reductase was 3x larger in OE than PF and controls. Circulating glucocorticoids changed little under OE, in spite of higher adrenal gland and liver content, hinting at modulation of glucocorticoid turnover as instrumental in their purported increased activity. In conclusion, we have observed that OE considerable enhanced the expression of the genes controlling the synthesis of glucocorticoids from cholesterol in the rat and increasing the adrenal glands' corticosterone, size and cellularity, but also the liver disposal of corticosteroids, suggesting that OE increases corticosterone synthesis and degradation (i.e. serum turnover), a process not driven by limited energy availability but directly related to the administration of OE.

The European sea bass is a teleost fish that lacks sex chromosomes and for which temperature influences sex ratios. However, correlation between temperature, developmental stage at a given age and sex-specific gene expression is hampered by the lack of sex markers. To study this correlation, fish were exposed to feminizing (15 degrees C) or masculinizing temperature (21 degrees C) from 0-120 days post fertilization, throughout the thermosensitive period (TSP). Aromatase (cyp19a1a), 11beta-hydroxylase (cyp11b), androgen receptor (arb) and estrogen receptors (era, erb1 and erb2) were assessed by qPCR prior and during sex differentiation. Canonical discriminant analysis (CDA), with length--as proxy for developmental stage--and cyp19a1a expression as predictors, was validated and used to reliably assign gonadal sex to fish sampled within and outside the TSP. Differences in cyp19a1a and cyp11b expression could be detected 1-month before the first signs of histological sex differentiation. Cyp19a1a and cyp11b were significantly higher in future females and males, respectively, and revealed as robust molecular markers to predict future ovarian and testicular differentiation. In contrast, no association between phenotypic sex and arb, era, erb1 and erb2 expression was found, suggesting that these genes do not contribute to the differentiation of a particular sex. The CDA-based approach implemented here could be used to sex undifferentiated animals in species where genetic sex cannot be known owing to the lack of simple sex determining systems, as it is the case of many fish and reptiles with or without temperature-dependent sex determination, and provide a useful tool to relate gene expression and phenotypic sex.

Excessive production of aldosterone has been implicated in the pathogenesis of hypertension and heart failure. One approach to ameliorate the deleterious effects of aldosterone is to suppress its biosynthesis. The enzyme aldosterone synthase (CYP11B2) is responsible for the final step of aldosterone synthesis. It requires electron transfer from the adrenodoxin/adrenodoxin reductase system to catalyze the production of aldosterone. A stable cell line simultaneously overexpressing recombinant human CYP11B2 as well as human adrenodoxin and adrenodoxin reductase was established to help maximize the enzyme activity. The homogenate of these cells was used to develop an in vitro CYP11B2 assay using 11-deoxycorticosterone as a substrate. By the same strategy, another stable cell line simultaneously overexpressing human 11beta-hydroxylase (CYP11B1), an enzyme responsible for the final step of cortisol biosynthesis, and the two electron transfer proteins was also established, and an in vitro CYP11B1 assay using 11-deoxycortisol as a substrate was likewise developed to assess the selectivity of CYP11B2 inhibitors. FAD286, a reference CYP11B2 inhibitor, inhibited CYP11B2 and CYP11B1 activities with IC(50) values of 1.6+/-0.1 and 9.9+/-0.9 nM (mean+/-SEM, n=3-6), respectively. Kinetics studies revealed that the compound inhibited the activity of both enzymes competitively with respective K(i) values of 0.8+/-0.04 and 2.2+/-0.2 nM (n=3-4). These assays can be used for assessing the potency and selectivity of CYP11B2 inhibitors for the treatment of hypertension and heart failure.

Steroids are known to play a crucial role in gonadal sex differentiation in many non-mammalian vertebrates, but also in the gonadal sex change of hermaphroditic teleosts. We investigated the expression of two genes encoding key steroidogenic enzymes, i.e., cytochrome P450 aromatase (P450arom) and cytochrome P45011beta-hydroxylase (P45011beta), during the sex change of the protogynous rice field eel, Monopterus albus. Using RT-PCR with degenerate primers, we cloned rice field eel homologous fragments for both genes (rcP450arom and rcP45011beta) as indicated by the high level of homology with P450arom and P45011beta sequences from various vertebrates. Gonadal expression of rcP450arom and rcP45011beta mRNA levels were then assessed during the sex change by semi-quantitative RT-PCR and a real-time RT-PCR. rcP450arom was predominantly expressed in ovary, much less in ovotestis, and barely in testis. Conversely, P45011beta was markedly up-regulated at the onset of testicular development. These findings underline that regulation of steroidogenesis is an important process in the sex change of protogynous rice field eel, and they clearly indicate that the concomitant down-regulation of P450arom and up-regulation of P45011beta are of pivotal importance to the sex change of this species.

At the GABA(A) receptor, low concentrations of etomidate potentiate the inhibitory effect of GABA on specific binding of the closed channel ligand [(3)H]ethynylpropylbicycloorthobenzoate ([(3)H]EBOB). Here, we present SARs for etomidate and structurally related compounds inducing this effect. In the absence of GABA, similar SARs, but 14-20 times weaker potencies were observed. We discuss these SARs in comparison to the much higher potencies of these compounds as inhibitors of 11beta-hydroxylase.

ILLUMINATE (Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events), the phase 3 morbidity and mortality trial of torcetrapib, a cholesteryl ester transfer protein (CETP) inhibitor, identified previously undescribed changes in plasma levels of potassium, sodium, bicarbonate, and aldosterone. A key question after this trial is whether the failure of torcetrapib was a result of CETP inhibition or of some other pharmacology of the molecule. The direct effects of torcetrapib and related molecules on adrenal steroid production were assessed in cell culture using the H295R as well as the newly developed HAC15 human adrenal carcinoma cell lines. Torcetrapib induced the synthesis of both aldosterone and cortisol in these two in vitro cell systems. Analysis of steroidogenic gene expression indicated that torcetrapib significantly induced the expression of CYP11B2 and CYP11B1, two enzymes in the last step of aldosterone and cortisol biosynthesis pathway, respectively. Transcription profiling indicated that torcetrapib and angiotensin II share overlapping pathways in regulating adrenal steroid biosynthesis. Hormone-induced steroid production is mainly mediated by two messengers, calcium and cAMP. An increase of intracellular calcium was observed after torcetrapib treatment, whereas cAMP was unchanged. Consistent with intracellular calcium being the key mediator of torcetrapib's effect in adrenal cells, calcium channel blockers completely blocked torcetrapib-induced corticoid release and calcium increase. A series of compounds structurally related to torcetrapib as well as structurally distinct compounds were profiled. The results indicate that the pressor and adrenal effects observed with torcetrapib and related molecules are independent of CETP inhibition.

The persistent environmental pollutant 3-methylsulfonyl-DDE (3-MeSO2-DDE) undergoes bioactivation by cytochrome P450 11B1 (CYP11B1) in the adrenal cortex of several animal species in vivo and causes decreased glucocorticoid production and cell death in the zona fasciculata. This study presents extended investigations of the cytotoxic and endocrine disrupting effects of 3-MeSO2-DDE and some structurally related molecules in the mouse adrenocortical cell line Y-1. Both 3-MeSO2-DDE and, to a lesser extent, 3,3'(bis)-MeSO2-DDE decreased corticosterone production and produced CYP11B1-dependent cytotoxicity in Y-1 cells. Neither 2-MeSO2-DDE nor p,p'-DDE had any significant effect on either cell viability or corticosterone production, indicating that the presence and position of the methylsulfonyl moiety of 3-MeSO2-DDE is crucial for its biological activity. The adrenocortical toxicant o,p'-DDD decreased corticosterone production but was not cytotoxic in this cell line. None of the compounds altered Cyp11b1 gene expression, indicating that 3-MeSO2-DDE inhibits CYP11B1 activity on the protein level.

Aldosterone is a key regulator of electrolyte and water homeostasis and plays a central role in blood pressure regulation. Hormonal changes during pregnancy, among them increased progesterone and aldosterone production, lead to the required plasma volume expansion of the maternal body as an accommodation mechanism for fetus growth. This review discusses the regulation of aldosterone production by aldosterone synthase (CYP11B2); the impact on aldosterone secretion due to the presence of a chimeric gene originating from a crossover between CYP11B1 and CYP11B2 in glucocorticoid remediable aldosteronism (GRA) - the inherited form of hypertension; enhanced aldosterone production in aldosterone-producing adenoma (APA); and idiopathic hyperaldosteronism (IHA). Features of hyperaldosteronism are also found in patients with apparent mineralocorticoid excess (AME), in which glucocorticoids exacerbate activation of the mineralocorticoid receptor (MR) because of a defect in the 11beta-hydroxysteroid dehydrogenase type 2 enzyme. Regulation of aldosterone production and tissue-specific activation of the mineralocorticoid receptor are prerequisites for optimal control of body fluids and blood pressure during pregnancy and contribute largely to the wellbeing of the mother-to-be.

Adrenocortical carcinoma is an uncommon malignancy and feminizing symptoms secondary to adrenal estrogen-secretion are extremely rare. The direct secretion of estradiol by adrenocortical tumors requires, in addition to the expression of aromatase (CYP19), the expression of one or more of the reductive 17beta-hydroxysteroid dehydrogenases. The expression of CYP19 transcripts and protein were markedly induced in the H295 adrenocortical carcinoma cell line after treatment with either forskolin or vasoactive intestinal peptide (VIP). Western immunoblotting demonstrated a marked induction of the CYP19 protein of characteristic size after only a short (6h) treatment period with VIP or forskolin. The CYP19 mRNA transcripts were derived from both promoters PII (Ic) and I.3 (Id) after treatment with both agents. The reductive type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3) was also constitutively expressed in the H295 cells but neither its mRNA transcript nor protein levels were altered after forskolin or VIP treatment. Western immunoblotting of an estrogen-secreting adrenal carcinoma revealed notable levels of both aromatase and AKR1C3 expression while an aldosterone-producing adrenal adenoma lacked aromatase expression and showed a reduced level of AKR1C3 expression. Immunohistochemistry of the carcinoma-bearing adrenal revealed localization of AKR1C3 not only in the tumor but also principally in the zona reticularis of the normal adrenal tissue. Adrenal aromatase and AKR1C3 expression therefore appear to be features of adrenocortical malignancies that are associated with biosynthesis of active estrogen.

Aldosterone plays an important role in electrolyte and blood pressure homeostasis. Our studies have focused on the role of aldosterone in essential hypertension. We have shown that plasma aldosterone and ARR are heritable characteristics and that aldosterone concentrations in older subjects are inversely correlated with birthweight and positively correlated with blood pressure. Aldosterone levels are also associated with polymorphic variation in the CYP11B2 gene, which encodes aldosterone synthase, the enzyme responsible for aldosterone production. Interestingly, CYP11B2 polymorphisms are also associated with less efficient activity of 11beta-hydroxylase, encoded by the neighbouring, highly homologous CYP11B1 gene. We propose that a digenic effect leads to increased aldosterone production, with inefficient 11beta-hydroxylation causing a long-term increase in ACTH drive to the adrenal gland and enhanced expression of CYP11B2, thereby resulting in chronically raised aldosterone secretion in response to factors such as angiotensin II and potassium. In susceptible subjects this is likely, over many years, to result in hypertension with relative aldosterone excess.

Blockade of a mineralocorticoid receptor is a clinically useful approach to the prevention of cardiovascular disease. The present study was designed to evaluate the effect of azelnidipine, a unique dihydropyridine Ca(2+) channel blocker, on aldosterone production in the human adrenocortical cell line NCI-H295R. Azelnidipine inhibited angiotensin II- and KCl-induced expression of steroid 11beta-hydroxylase, steroid 18-hydroxylase, and the alpha1H subunit of the T-type Ca(2+) channel, and suppressed steroid biosynthesis in H295R cells by the same amount as efonidipine. On the basis of these findings, azelnidipine appears to suppress steroid biosynthesis in H295R cells beyond the blockade of L-type calcium channels.

Steroidogenic factor-1 (SF-1/Ad4BP/NR5A1) plays a major role in regulating steroidogenic enzymes. We have previously shown that SF-1 inhibits aldosterone synthase (CYP11B2) reporter gene activity. Herein, we used the H295R/TR/SF-1 adrenal cells that increase SF-1 in a doxycycline-dependent fashion. Cells were incubated with or without doxycycline to induce SF-1 and then treated with angiotensin II (Ang II). Aldosterone was measured by immunoassay. SF-1 mRNA was silenced by small interfering RNA (siRNA) by Nucleofector technology. mRNA levels were measured by real-time RT-PCR. Ang II treatment without doxycycline increased aldosterone production by 11.3-fold and CYP11B2 mRNA by 116-fold. Doxycycline treatment increased SF-1 mRNA levels by 3.7-fold and inhibited Ang II-induced aldosterone by 84%. Doxycycline treatment inhibited Ang II-stimulated CYP11B2 mRNA levels by 86%. Doxycycline decreased basal CYP11B2 promoter activity by 68%. Doxycycline inhibited Ang II stimulation by 85%. Ang II increased CYP21 mRNA expression by 4.6-fold, whereas doxycycline inhibited induction by 69%. In contrast, doxycycline treatment increased CYP11B1 mRNA by 1.7-fold in basal cells and increased Ang II induction by 3.6-fold. SF-1-specific siRNA significantly reduced SF-1 mRNA expression as compared with cells treated with control siRNA. SF-1 siRNA reversed doxycycline stimulation of CYP B1 and its inhibition of CYP11B2. However, in H295R/TR/SF-1 cells without doxycycline treatment, both CYP11B1 and CYP11B2 mRNAs were significantly decreased, suggesting that both enzymes require a minimal level of SF-1 for basal expression. In summary, SF-1 overexpression dramatically inhibited CYP11B2 expression and decreased aldosterone production. The opposing effects of SF-1 on CYP11B1 and CYP11B2 suggest that the regulation of SF-1 activity may play a role that determines the relative ability to produce mineralocorticoid and glucocorticoid.

Synchronous associations of Cushing's syndrome (CS) and primary aldosteronism (PA) with multiple adrenocortical adenomas secreting each hormone independently have rarely been reported. Herein, we describe a unique case of PA associated with CS with detailed clinical and pathological investigations. Bilateral adrenal masses with clinical symptoms of CS and PA were found in a 43-year-old woman. Venous sampling demonstrated excess secretion of cortisol, and aldosterone from right, and left tumor, respectively. A bilateral laparoscopic partial adrenalectomy was undergone. The right adrenal tumor (3 cm) was yellow in color with abundant lipofuscin granules, and was composed of both eosinophilic compact cells and clear cells. In situ hybridization showed that both mRNAs for HSD3B2 and CYP17A1 were strongly expressed in the tumor, suggesting cortisol synthesis. Left adrenal tumor (2.4 cm) was golden-yellow in color, and composed of clear cells only. Expression of HSD3B2 and CYP11B mRNAs were observed in the tumor compatible with the aldosterone synthesis. Furthermore, minute nodules were found at the surface of normal-appearing cortex on both sides of the adrenal glands, and the expression of HSD3B2 and CYP11B mRNAs was clearly demonstrated within the nodules, indicating aldosterone synthesis. We diagnosed that the present case had 1) cortisol-producing right adrenocortical adenoma, 2) aldosterone producing left adrenocortical adenoma, and 3) cortical minute nodules with aldosterone production in both adrenal glands compatible with idiopathic adrenal hyperplasia. We reviewed the cases reported, and discussed the significance of the minute nodules in the adrenal cortex, often found in association with the adrenocortical adenoma.

Objective: To investigate the effects of Semen descurainiae and Captopril on CYP11B1, CYP11B2 and TGF-beta1 mRNA expression of heart tissue in rats treated with Abdominal Aortic Banding., Methods: Ventricular remodeling was induced by abdominal aortic banding (AAB) in rats. After 30 days' treatment, the ratios of LVW/BW (left ventricle weight/body weight), HW/BW (heart weight/body weight) were calculated; Then the CYP11B, CYP11B2 and TGF-beta1 mRNA expression of left ventricle were detected by Real-time PCR, respectively., Results: The experimental data demonstrated that Semen descurainiae decreased the indexes of LVW/BW and HW/BW, down-regulated CYP11B, CYP11B2 and TGF-beta1 mRNA expression in left ventricle (P<0.05)., Conclusion: Semen desceurainiae can significantly inhibit the experimental ventricular remodeling; the mechanism is related to its ability to attenuate the mRNA expression of CYP11B1, CYP11B2 and TGF-beta1 in left ventricle. The inhibition of aldosterone key gene expression by Semen descurainiae may contribute to its effect on restraint cardiac remodeling.

The role of genes implicated in the regulation of spermatogenesis and their patterns of expression is still poorly understood. In this study, we took advantage of the cystic arrangement of the teleost testis to set up a laser capture microdissection procedure to isolate cells from cysts containing spermatogonia, spermatocytes, spermatids, or spermatozoa. We then used quantitative PCR to determine the stage-specific expression patterns of the germ cell marker vasa; gonadal aromatase (cyp19a); estrogen receptors (ers) alpha, beta1, and beta2 (era, erb1, and erb2, respectively); 11beta-hydroxylase (cyp11b1); androgen receptor beta (arb); insulinlike growth factor 1 (igf1); and sox17. vasa had the highest mRNA levels, followed by genes involved in androgen metabolism (cyp11b1 and arb). Most genes associated with estrogen metabolism (cyp19a, era, and erb1) had a lower expression, whereas igf1 and sox17 exhibited the lowest mRNA levels. Comparison of changes in mRNA levels revealed five patterns of gene expression, in general with progressively lower expression seen as spermatogenesis advanced. igf1 and sox17 were exclusively expressed in spermatogonia-containing cysts, suggesting effects during the proliferative stage. Genes involved in androgen synthesis (cyp11b1) and action (arb) peaked during the early stages of spermatogenesis and then sharply decreased. In contrast, genes associated with estrogen action, particularly erb2 and era, showed a more gradual decrease. Together, these results demonstrate the usefulness of fish models and suggest that whereas androgens are required at high levels and may exert their major actions at the initial stages of spermatogenesis, estrogens are also essential, albeit required at lower levels, and with a more generalized influence.

Objective: It is unknown why some patients with biochemical evidence of primary aldosteronism (PA) do not develop hypertension. We aimed to compare clinical and biochemical characteristics of normotensive and hypertensive patients with PA., Design and Patients: Retrospective comparison of 10 normotensive and 168 hypertensive patients with PA for office or ambulatory blood pressure, serum potassium, plasma aldosterone and renin concentrations; the aldosterone:renin ratio, and tumour size. Comparison of initial hormonal pattern and drop in blood pressure following adrenalectomy in five normotensive and nine hypertensive patients matched for age, sex and body mass index., Results: The 10 normotensive patients were women and presented with hypokalemia or an adrenal mass. Age, plasma aldosterone and renin concentrations were similar in normotensive and hypertensive cases, but kalemia and body mass index were significantly lower in the normotensive patients. Mean tumour diameter was larger in the normotensive patients than in the hypertensive matched patients with an adenoma (P < 0.01). In normotensive patients, diastolic blood pressure and upright aldosterone correlated negatively with kalemia. Blood pressure was lowered similarly after adrenalectomy in five normotensive PA patients and in their matched hypertensive counterparts. Aldosterone synthase expression was detected in four out of five adrenal tumours., Conclusions: Blood pressure may be normal in patients with well-documented PA. The occurrence of hypokalemia, despite a normal blood pressure profile, suggests that protective mechanisms against hypertension are present in normotensive patients.

Background: Congenital adrenal hyperplasia (CAH), due to deficiency in 11â hydroxylase enzyme is rare a cause of virilization associated with glucocorticoid responsive hypertension., Objective: To present a rare cause of CAH and secondary hypertension responsive to glucocorticoid therapy., Methods: Clinical and laboratory evaluation of a young woman with a "phallus" and hypertension. Investigations carried out included serum biochemistry, bone age determination, sex chromatin evaluation and serum levels of androgens and 17-OH progesterone, as well as pelvic ultrasonography., Results: There was a history of cliteromegaly, first noticed at age 14 years, but menstrual periods were said to be regular. Physical findings included male type hairline and musculature with a blood pressure of 160/110mmHg. Breast development was Tanner Grade 3 while the public hair was male in pattern. The clitoris was 5cm long. Serum electrolytes were normal but levels of testosterone, dehydroepiandosterone sulphate and 17-OH progesterone were elevated. Features were compatible with a diagnosis of congenital adrenal hyperplasia (CAH) due to Il-beta-hydroxylase defficiency. Hypertension responded to glucocorticoid therapy, recurred on default and was corrected again on re-starting hydrocortisone., Conclusion: Congenital adrenal hyperplasia due to 11â-hydroxylase deficiency though rare, should be considered in the differential diagnosis of hypertension with virilization in young females.

Benidipine, a long-lasting dihydropyridine calcium channel blocker, is used for treatment of hypertension and angina. Benidipine exerts pleiotropic pharmacological features, such as renoprotective and cardioprotective effects. In pathophysiological conditions, the antidiuretic hormone aldosterone causes development of renal and cardiovascular diseases. In adrenal glomerulosa cells, aldosterone is produced in response to extracellular potassium, which is mainly mediated by T-type voltage-dependent Ca2+ channels. More recently, it has been demonstrated that benidipine inhibits T-type Ca2+ channels in addition to L-type Ca2+ channels. Therefore, effect of calcium channel blockers, including benidipine, on aldosterone production and T-type Ca2+ channels using human adrenocortical cell line NCI-H295R was investigated. Benidipine efficiently inhibited KCl-induced aldosterone production at low concentration (3 and 10 nM), with inhibitory activity more potent than other calcium channel blockers. Patch clamp analysis indicated that benidipine concentration-dependently inhibited T-type Ca2+ currents at 10, 100 and 1000 nM. As for examined calcium channel blockers, inhibitory activity for T-type Ca2+ currents was well correlated with aldosterone production. L-type specific calcium channel blockers calciseptine and nifedipine showed no effect in both assays. These results indicate that inhibition of T-type Ca2+ channels is responsible for inhibition of aldosterone production in NCI-H295R cells. Benidipine efficiently inhibited KCl-induced upregulation of 11-beta-hydroxylase mRNA and aldosterone synthase mRNA as well as KCl-induced Ca2+ influx, indicating it as the most likely inhibition mechanism. Benidipine partially inhibited angiotensin II-induced aldosterone production, plus showed additive effects when used in combination with the angiotensin II type I receptor blocker valsartan. Benidipine also partially inhibited angiotensin II-induced upregulation of the above mRNAs and Ca2+ influx inhibitory activities of benidipine for aldosterone production. T-type Ca2+ channels may contribute to additional benefits of this drug for treating renal and cardiovascular diseases, beyond its primary anti-hypertensive effects from blocking L-type Ca2+ channels.

Derivatives of etomidate were evaluated as inhibitors of adrenal steroid 11beta-hydroxylations. Stereoselective coupling by Mitsunobu produced chirally pure analogues to study the effect of configuration, modification of the ester, and substitution in the phenyl ring, with the aim to probe specific sites for introducing a radionuclide. Iodophenyl metomidate (IMTO) labeled with iodine-131 served as radioligand for structure-affinity relationship studies. We have characterized the kinetic parameters of specific (131)I-IMTO binding on rat adrenal membranes and used the displacement of (131)I-IMTO binding to evaluate functionalized MTO analogues. Our results indicated that (1) ( R)-configuration is essential for high affinity, (2) highest potency resides in the ethyl, 2-propyl, and 2-fluoroethyl esters, and (3) substitution of the phenyl ring is well tolerated. The clinically used inhibitors metyrapone and ketoconazole inhibited (131)I-IMTO binding with low affinity. Incubation of selected analogues with human adrenocortical NCI-h295 cells demonstrated a high correlation with the inhibitory effect on cortisol secretion.

Morphine withdrawal leads to an increase in corticosterone concentration in plasma, and cognitive deficits are found after withdrawal. Evidence indicates that glucocorticoid hormones affect memory. The aim of the current study was to evaluate the effects of metyrapone and mifepristone on memory deficit following spontaneous morphine withdrawal. Memory was tested by using the object recognition task. The novel object recognition task was carried out in a square wooden open-field apparatus using objects. The test was comprised of three sections: habituation for 15 min., first trial for 12 min. and test trial for 5 min. In this learning paradigm, the difference in exploration between a previously seen object and a novel object is taken as an index of memory performance (recognition index - RI). Male mice were made dependent by increasing doses of morphine (30-90 mg/kg) subcutaneously twice daily for 3 days. RI was assessed 4 hr after the last dose of morphine on the third day. Mifepristone (50 and 100 mg/kg) and metyrapone (12.5 and 25 mg/kg) were used subcutaneously before the first trial and effects were compared to control values. Metyrapone (25 mg/kg) and mifepristone (50 mg/kg) improved RI to 34.8 +/- 10.8% and 25.4 +/- 11.7%, respectively, which are significantly different from control values (RI = -14.8 +/- 10.7%, P < 0.05). These results show that increased glucocorticoid concentration may be involved in memory deficit caused by morphine withdrawal. Metyrapone by inhibiting glucocorticoid formation, and mifepristone by inhibiting glucocorticoid receptors may be useful for preventing memory deficit following morphine withdrawal.

Congenital adrenal hyperplasia due to steroid 11beta-hydroxylase deficiency is a genetic disorder of steroidogenesis, transmitted as an autosomal recessive trait. It is associated with low renin hypertension, hypokalemia, hyperandrogenemia and genital ambiguity in affected females. Mutations in the CYP11B1 gene, causing 11beta-hydroxylase deficiency in the zona fasciculata in the adrenal cortex, have been identified. The indicators of congenital adrenal hyperplasia caused by 11beta-hydroxylase deficiency, include increased serum concentrations of desoxycorticosterone, 11 deoxycortisol and delta4-androstenedione, and suppressed plasma renin concentrations. The disorder is treated by administration of glucocorticoids.

Using a highly sensitive quantitative RT-PCR method for the measurement of CYP11B1 (11beta-hydroxylase) and CYP11B2 (aldosterone synthase) mRNAs, we previously demonstrated that CYP11B2 expression in the central nervous system (CNS) is subject to regulation by dietary sodium. We have now quantified the expression of these genes in the CNS of male Wistar Kyoto (WKY) rats in response to systemic ACTH infusion, dexamethasone infusion, and to adrenalectomy. CYP11B1 and CYP11B2 mRNA levels were measured in total RNA isolated from the adrenal gland and discrete brain regions using real-time quantitative RT-PCR. ACTH infusion (40 ng/day for 7 days, N=8) significantly increased CYP11B1 mRNA in the adrenal gland, hypothalamus, and cerebral cortex compared with animals infused with vehicle only. ACTH infusion decreased adrenal CYP11B2 expression but increased expression in all of the CNS regions except the cortex. Dexamethasone (10 microg/day for 7 days, N=8) reduced adrenal CYP11B1 mRNA compared with control animals but had no significant effect on either gene's expression in the CNS. Adrenalectomy (N=6 per group) significantly increased CYP11B1 expression in the hippocampus and hypothalamus and raised CYP11B2 expression in the cerebellum relative to sham-operated animals. This study confirms the transcription of CYP11B1 and CYP11B2 throughout the CNS and demonstrates that gene transcription is subject to differential regulation by ACTH and circulating corticosteroid levels.

Context: The diagnosis of the polycystic ovary syndrome requires the exclusion of nonclassical congenital adrenal hyperplasia (NCAH)., Objective: Our objective was to evaluate the actual prevalences of 21-hydroxylase and 11beta-hydroxylase deficiencies among women presenting with hyperandrogenic complaints., Settings: This study was performed at an academic hospital., Patients: A total of 270 consecutive unselected women presenting with hyperandrogenic symptoms were prospectively recruited., Interventions: Basal and ACTH-stimulated 11-deoxycortisol and 17-hydroxyprogesterone concentrations were measured., Main Outcome Measures: The prevalences of 21-hydroxylase and 11beta-hydroxylase deficiencies were calculated, and the diagnostic performance of basal serum 17-hydroxyprogesterone levels for the screening of NCAH was evaluated by receiver operating characteristic curve analysis., Results: Six of the 270 patients had 21-hydroxylase-deficient NCAH that was confirmed by CYP21 genotyping, whereas no patient was diagnosed with 11beta-hydroxylase deficiency, for an overall NCAH prevalence of 2.2% (95% confidence limits 0.5-3.9%). According to receiver operating characteristic analysis, a single basal serum 17-hydroxyprogesterone determination has a 0.97 (95% confidence interval: 0.934-1.008) chance of detecting NCAH in hyperandrogenic women. In our experience, the most appropriate cutoff value for the detection of NCAH is a 17-hydroxyprogesterone above 1.7 ng/ml, showing a 100% sensitivity and a 88.6% specificity. Five of the six 21-hydroxylase-deficient NCAH patients carried a severe CYP21 allele requiring genetic counseling and highlighting the importance of excluding this disorder among hyperandrogenic patients., Conclusions: The prevalence of NCAH among hyperandrogenic patients from Spain is 2.2%. Basal serum 17-hydroxyprogesterone measurements have an excellent diagnostic performance, yet the cutoff value should be established in each laboratory to avoid false-negative results.

To obtain basic information on the endocrine mechanisms underlying sex change in the protandrous anemonefish Amphiprion clarkii, we examined the immunolocalization of the steroidogenic enzyme cytochrome 11beta-hydroxylase (P45011beta), which is involved in 11-ketotestosterone (11-KT) production, and analyzed the ability of gonads to produce steroid hormones throughout the sex differentiation process and at the breeding stage. Immunopositive reactions against P45011beta appeared in sexually undifferentiated gonads at 30 days post hatching (dph). The number of immunopositive cells continued to increase during ovarian differentiation (from 60 to 180 dph) and throughout the formation of ambisexual gonads with both ovarian and testicular tissue until 270 dph. In the male phase, strongly immunopositive cells were observed in the cellular interstices of both testicular and ovarian tissues. P45011beta was localized only in the theca cells enclosing developed oocytes in the female phase. In-vitro 11-KT production in the gonads gradually increased with testicular differentiation (before, during, and after differentiation). Production of 11-KT in the gonads was higher in the male phase than during testicular differentiation or in the female phase. Our results suggest that androgen is involved in testicular differentiation during sex differentiation and spermatogenesis.

The human 11beta-hydroxylase (hCYP11B1) is responsible for the conversion of 11-deoxycortisol into the major mammalian glucocorticoid, cortisol. The reduction equivalents needed for this reaction are provided via a short electron transfer chain consisting of a [2Fe-2S] ferredoxin and a FAD-containing reductase. On the biochemical and biophysical level, little is known about hCYP11B1 because it is very unstable for analyses performed in vitro. This instability is also the reason why it has not been possible to stably express it so far in Escherichia coli and subsequently purify it. In the present study, we report on the successful and reproducible purification of recombinant hCYP11B1 coexpressed with molecular chaperones GroES/GroEL in E. coli. The protein was highly purified to apparent homogeneity, as observed by SDS/PAGE. Upon mass spectrometry, the mass-to-charge ratio (m/z) of the protein was estimated to be 55 761, which is consistent with the value 55 760.76 calculated for the form lacking the translational initiator Met. The functionality of hCYP11B1 was analyzed using different methods (substrate conversion assays, stopped-flow, Biacore). The results clearly demonstrate that the enzyme is capable of hydroxylating its substrates at position 11-beta. Moreover, the determined NADPH coupling percentage for the hCYP11B1 catalyzed reactions using either 11-deoxycortisol or 11-deoxycorticosterone as substrates was approximately 75% in both cases. Biacore and stopped-flow measurements indicate that hCYP11B1 possesses more than one binding site for its redox partner adrenodoxin, possibly resulting in the formation of more than one productive complexes. In addition, we performed CD measurements to obtain information about the structure of hCYP11B1.

Cytochromes P450 play a vital role in the steroid biosynthesis pathway of the adrenal gland. An example of an essential P450 cytochrome is the steroid 11beta-hydroxylase CYP11B1, which catalyses the conversion of 11-deoxycorticol to hydrocortisone. However, despite its high biotechnological potential, this enzyme has so far been unsuccessfully employed in present-day biotechnology due to a poor expression yield and inherent protein instability. In this study, CYP11B1 was biotransformed into various strains of the yeast Schizosaccharomyces pombe, all of which also expressed the electron transfer proteins adrenodoxin and/or adrenodoxin reductase - central components of the mitochondrial P450 system - in order to maximise hydrocortisone production efficiency in our proposed model system. Site-directed mutagenesis of CYP11B1 at positions 52 and 78 was performed in order to evaluate the impact of altering the amino acids at these sites. It was found that the presence of an isoleucine at position 78 conferred the highest 11beta-hydroxylation activity of CYP11B1. Coexpression of adrenodoxin and adrenodoxin reductase appeared to further increase the 11beta-hydroxylase activity of the enzyme (3.4 fold). Adrenodoxin mutants which were found to significantly enhance enzyme efficiency in other cytochromes in previous studies were also tested in our system. It was found that, in this case, the wild type adrenodoxin was more efficient. The new fission yeast strain TH75 coexpressing the wild type Adx and AdR displays high hydrocortisone production efficiency at an average of 1mM hydrocortisone over a period of 72h, the highest value published to date for this biotransformation. Finally, our research shows that pTH2 is an ideal plasmid for the coexpression of the mitochondrial electron transfer counterparts, adrenodoxin and adrenodoxin reductase, in Schizosaccharomyces pombe, and so could serve as a convenient tool for future biotechnological applications.

Background/aim: 21-Hydroxylase deficiency congenital adrenal hyperplasia (CAH) is one of the target diseases in many newborn screening programs. 11beta-Hydroxylase defiency is less frequent and does not cause salt-losing crisis. Thus, it is not a target disease for newborn screening. However, affected newborns might show slightly elevated levels of 17-OH-progesterone (17-OHP) in the standard immunoassay screening test. The objective is to show that the diagnosis of 11beta-hydroxylase deficiency can be done using a dried blood spot from newborn screening., Case Report: A male newborn was born at term. Blood sample for newborn screening was taken 36 h after birth. 17-OHP was slightly elevated using time-resolved fluorescence immunoassay (72.8 nmol/l; cut-off <60 nmol/l)., Results: We performed second-tier LC-MS/MS from the same blood sample and found elevated levels of 11-deoxycortisol and androstenedione and low cortisol. The family history was positive with an affected older sister born with ambiguous genitalia. Confirmation of diagnosis was done by hormonal analysis and molecular genetic testing of the CYP11B1 gene. A known CYP11B1 gene mutation W116C was identified in this family., Conclusions: The diagnosis of 11beta-hydroxylase deficiency can be made by second-tier LC-MS/MS from dried blood spots. This method is very helpful in the work-up of elevated immunoassay 17-OHP., ((c) 2007 S. Karger AG, Basel)

Myopathy is a well-recognized complication of persistent hypokalemia. Although hypokalemic myopathy may be due to divers diseases or to drug administration, hypokalemic rhabdomyolysis as a complication of under-treated 11-hydroxylase deficiency has not previously been described in the literature. We describe a 10-year-old boy with under-treated 11-hydroxylase deficiency who developed rhabdomyolysis following severe hypokalemia. Patients with under-treated 11-hydroxylase deficiency may present with hypokalemia in association with muscle weakness; if serum potassium is markedly low, rhabdomyolysis may occur. Hypokalemia-induced rhabdomyolysis should be carefully followed.

Background/aim: 11-beta-hydroxylase deficiency (11betaOHD) is caused by CYP11B1 gene defects and leads to congenital adrenal hyperplasia associated with hypertension. Recently, a novel L299P mutation has been described in a compound heterozygous male individual. We observed two 46,XX siblings with a homozygous L299P mutation and investigated the functional properties of this CYP11B1 variant., Patients: The index patient from a consanguineous Turkish family showed complete external virilization and was diagnosed incidentally at the age of 19 months during hospital admission for severe combined bacterial (urosepsis) and viral (CMV and EBV) infection. The younger sibling was diagnosed at the age of 5 months. Their genital phenotype was identical and both demonstrated borderline elevated blood pressure., Results: Biochemical findings revealed 11betaOHD. A homozygous L299P mutation of the CYP11B1 gene was detected. In vitro expression studies performed in HCT116 cells showed a markedly decreased CYP11B1 activity in the L299P mutant (1.6 +/- 0.8%) for the conversion of 11-deoxycortisol to cortisol., Conclusions: Our study provides clear data on the functional properties and clinical phenotype in 46,XX individuals homozygous for this point mutation. Adrenal insufficiency might have contributed to the severe infectious disease that was present in the index patient at diagnosis., (Copyright 2008 S. Karger AG, Basel.)