Adrenal Zona Glomerulosa Research Articles

Functional TASK-3-Like Channels in Mitochondria of Aldosterone-Producing Zona Glomerulosa Cells.

Ca2+ drives aldosterone synthesis in the cytosolic and mitochondrial compartments of the adrenal zona glomerulosa cell. Membrane potential across each of these compartments regulates the amplitude of the Ca2+ signal; yet, only plasma membrane ion channels and their role in regulating cell membrane potential have garnered investigative attention as pathological causes of human hyperaldosteronism. Previously, we reported that genetic deletion of TASK-3 channels (tandem pore domain acid-sensitive K+ channels) from mice produces aldosterone excess in the absence of a change in the cell membrane potential of zona glomerulosa cells. Here, we report using yeast 2-hybrid, immunoprecipitation, and electron microscopic analyses that TASK-3 channels are resident in mitochondria, where they regulate mitochondrial morphology, mitochondrial membrane potential, and aldosterone production. This study provides proof of principle that mitochondrial K+ channels, by modulating inner mitochondrial membrane morphology and mitochondrial membrane potential, have the ability to play a pathological role in aldosterone dysregulation in steroidogenic cells.

Physiological and Pathological Roles in Human Adrenal of the Glomeruli-Defining Matrix Protein NPNT (Nephronectin).

Primary aldosteronism is a common cause of hypertension, which becomes refractory if undiagnosed, but potentially curable when caused by an aldosterone-producing adenoma (APA). The discovery of somatic mutations and differences in clinical presentations led to recognition of small but common zona glomerulosa (ZG)-like adenomas, distinct from classical large zona fasciculata-like adenomas. The inverse correlation between APA size and aldosterone synthase expression prompted us to undertake a systematic study of genotype-phenotype relationships. After a microarray comparing tumor subtypes, in which NPNT (nephronectin) was the most highly (>12-fold) upregulated gene in ZG-like APAs, we aimed to determine its role in physiological and pathological aldosterone production. NPNT was identified by immunohistochemistry as a secreted matrix protein expressed exclusively around aldosterone-producing glomeruli in normal adrenal ZG and in aldosterone-dense ZG-like APAs; the highest expression was in ZG-like APAs with gain-of-function CTNNB1 mutations, whose removal cured hypertension in our patients. NPNT was absent from normal zona fasciculata, zona fasciculata-like APAs, and ZG adjacent to an APA. NPNT production was regulated by canonical Wnt pathway, and NPNT overexpression or silencing increased or reduced aldosterone, respectively. NPNT was proadhesive in primary adrenal and APA cells but antiadhesive and antiapoptotic in immortalized adrenocortical cells. The discovery of NPNT in the adrenal helped recognition of a common subtype of APAs and a pathway by which Wnt regulates aldosterone production. We propose that this arises through NPNT's binding to cell-surface integrins, stimulating cell-cell contact within glomeruli, which define ZG. Therefore, NPNT or its cognate integrin could present a novel therapeutic target.

Very low-density lipoprotein (VLDL)-induced signals mediating aldosterone production.

Aldosterone, secreted by the adrenal zona glomerulosa, enhances sodium retention, thus increasing blood volume and pressure. Excessive production of aldosterone results in high blood pressure and contributes to cardiovascular and renal disease, stroke and visual loss. Hypertension is also associated with obesity, which is correlated with other serious health risks as well. Although weight gain is associated with increased blood pressure, the mechanism by which excess fat deposits increase blood pressure remains unclear. Several studies have suggested that aldosterone levels are elevated with obesity and may represent a link between obesity and hypertension. In addition to hypertension, obese patients typically have dyslipidemia, including elevated serum levels of very low-density lipoprotein (VLDL). VLDL, which functions to transport triglycerides from the liver to peripheral tissues, has been demonstrated to stimulate aldosterone production. Recent studies suggest that the signaling pathways activated by VLDL are similar to those utilized by AngII. Thus, VLDL increases cytosolic calcium levels and stimulates phospholipase D (PLD) activity to result in the induction of steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2) expression. These effects seem to be mediated by the ability of VLDL to increase the phosphorylation (activation) of their regulatory transcription factors, such as the cAMP response element-binding (CREB) protein family of transcription factors. Thus, research into the pathways by which VLDL stimulates aldosterone production may identify novel targets for the development of therapies for the treatment of hypertension, particularly those associated with obesity, and other aldosterone-modulated pathologies.

Effects of chronic administration of the phosphodiesterase inhibitor Vardenafil on serum levels of adrenal and testicular steroids in men with type 2 diabetes mellitus

Background. Steroidogenesis is a complex enzymatic process in which cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) play an important role. Phosphodiesterase-5 inhibitors(PDE5i) increase cGMP, improving NO availability.Objective. to investigate whether long-term, chronic treatment with the PDE5i Vardenafil affects adrenal and testicular steroidogenesis in diabetic men, using liquid chromatography-mass spectrometry (LC-MS/MS).Design. A longitudinal, prospective, investigator-started, randomized, placebo-controlled, double-blind, clinical-trial was carried out.Setting and Participants. 54 male patients affected by T2DM diagnosed within the last 5 years were enrolled. 26 and 28 patients were assigned to the verum and placebo-group, respectively.Interventions. The study consisted of an enrolment phase, a treatment phase (24weeks) (Vardenafil/placebo 10mg twice-daily), and a follow-up phase (24weeks).Outcome measurements: progesterone (P), 17-hydroxyprogesterone (17OHP), androstenedione (A), testosterone (T), dehydroepiandrosterone (DHEA), DHEA sulphate (DHEAS), corticosterone, 11-deoxycortisol and cortisol (C), were evaluated using LC-MS/MS.Results. No differences were seen in sex testicular steroids between study and control group. For the adrenal gland, steroids were considered according to the zona in which they are produced. Considering steroids produced in the zona fasciculata, no significant differences were seen in 11-deoxycortisol and C among visits, both in the study and in the control group. For the zona reticularis, DHEA significantly decreased during treatment only in the study group (p=0.007). At post-hoc test DHEA showed higher levels at visit 2 and 8 than in other visits. The DHEAS/DHEAS ratio significantly increased during treatment only in the verum group. Considering the adrenal zona glomerulosa, corticosterone significantly changed among visits both in the study and in the control group (p<0.001). At post-hoc test, in both groups, corticosterone was significantly higher at visit 2 (p=0.028), 8 (p=0.003), and 10 (p=0.044), i.e. in coincidence with the complete clinical and instrumental examination performed only at these visits according to the study protocol.Conclusions. This is the first double-blind, placebo-controlled clinical-trial in which steroidogenesis is extensively investigated by LC-MS/MS in T2DM men chronically treated with Vardenafil for 6 months, and followed-up for 6 months after therapy-withdrawal. Chronically administered Vardenafil reduces DHEA levels and increases DHEAS/DHEA ratio as possible consequences of modulation of steroidogenic enzymes by tissue changes in cGMP and/or cAMP availability. A possibly stress-related increase in corticosterone is suggested for the first time.

Abstract 097: Effects of Lysine-specific Demethylase 1 Deficiency on Aldosterone Production and Salt-sensitive Hypertension in Female Mice

Lysine-Specific Demethylase1 (LSD1) is an epigenetic factor modulated by salt intake. Previously, we documented the male heterozygote LSD1 knockout mice (LSD1+/-) had dysregulation of aldosterone (ALDO) production on a liberal salt diet (1.6% Na+) associated with salt-sensitive hypertension. This study assessed if: 1) female LSD1+/- mice have a similar phenotype; and 2) the effect of aging on this phenotype. Methods: Female LSD1+/- and wild type mice (LSD1+/+) were randomly assigned for sacrifice at the ages of 18-week, 52-week, and 75-week and the following were assessed at each time point: blood pressure (BP); plasma renin activity (PRA) and ALDO; urine albumin; and ex vivo ALDO production from isolated adrenal zona glomerulosa cells. Results: BP and urine albumin in the LSD1+/- compared to the LSD1+/+ were not different at any age (Table). However, the LSD1+/- had greater ALDO/PRA ratios at 18 weeks compared with the LSD1+/+, but lower ALDO levels and ex vivo ALDO production at 52 and 75 weeks. Associated with this phenotype, the LSD1+/- showed significantly higher rate of all-cause mortality than the LSD1+/+. Conclusion: Lack of LSD1 caused dysregulation of ALDO production in both male and female mice. But the cardiovascular outcomes are different. The LSD1+/- females in contrast to males do not develop hypertension or albuminuria even at 75 weeks of age. However, the females do die at a faster rate than the males of a variety of causes. Thus, there is considerable sexual dimorphism in the pathogenesis of cardiovascular outcomes associated with dysregulation of adrenal ALDO production mediated by lack of LSD1.

Abstract 128: Leptin: A Regulator of Aldosterone Synthase Expression & Aldosterone Secretion in Visceral Adipocytes, in Mice

Obesity is associated with inappropriately high aldosterone levels, which contribute to the development of metabolic and cardiovascular disorders. The origin of these high aldosterone levels is incompletely understood. We recently demonstrated that the adipocyte-derived hormone leptin regulates aldosterone synthase (CYP11B2) expression and stimulates aldosterone release from adrenal zona glomerulosa cells. Recent studies demonstrate that adipocytes express CYP11B2 and secrete aldosterone. However, the mechanisms regulating aldosterone release from adipocytes remain unclear. Likewise, whether visceral (Visc) and subcutaneous (SubQ) adipose tissue contribute to a similar extent to aldosterone production is unknown. We tested the hypothesis that leptin increases adipocyte CYP11B2 expression and aldosterone production and investigated whether Visc and SubQ adipose tissues respond similarly to leptin. Immunostaining of mouse adipose tissue cross-sections and isolated mature adipocytes revealed that Visc and SubQ adipose tissue express leptin receptors. Treatment of mouse freshly isolated mature adipocytes, non-differenciated (stromal fraction) and differentiated adipocytes revealed that leptin dose-dependently increased CYP11B2 expression and aldosterone production in Visc adipose tissue only. Although leptin receptor and CYP11B2 levels were similar in SubQ and Visc adipocytes, SubQ adipocytes were unresponsive to leptin. The physiological relevance of these in vitro data was tested by measuring plasma aldosterone levels in mice deprived of adipose tissue (lipodystrophic mice) treated with leptin. Absence of adipose tissue in lipodystrophic mice blunted leptin-induced increases in aldosterone levels (WT-vehicle: 471±82 vs. WT-Leptin: 1699±396, p<0.05; KO-vehicle: 539±71 vs. KO+leptin: 787±156, NS). The human relevance of these data was determined by reporting that CYP11B2 expression gradually increased with body mass index in human mediastinal and omental fat depots. In summary these data strongly suggest that leptin regulates CYP11B2 levels and aldosterone release in visceral adipose tissue and that leptin-induced, adipocyte-derived aldosterone may contribute to obesity-associated hyperaldosteronism.

Effects of chronic administration of the phosphodiesterase inhibitor vardenafil on serum levels of adrenal and testicular steroids in men with type 2 diabetes mellitus.

To investigate whether long-term, chronic treatment with the phosphodiesterase-5 inhibitor vardenafil affects adrenal and testicular steroidogenesis in diabetic men, using liquid chromatography-tandem mass spectrometry. A longitudinal, prospective, investigator-started, randomized, placebo-controlled, double-blind, clinical-trial was carried out, enrolling 54 male patients affected by type 2 diabetes mellitus diagnosed within the last 5 years. In total, 26 and 28 patients were followed for 1 year and assigned to the study and placebo group, respectively. Progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, corticosterone, 11-deoxycortisol and cortisol, were evaluated using liquid chromatography-tandem mass spectrometry. No differences were seen in sex testicular steroids between study and control group. As for the adrenal gland, steroids were considered according to the zona in which they are produced. No significant differences were seen in steroid produced in zona fasciculata. For the zona reticularis, dehydroepiandrosterone significantly decreased during treatment only in the study group (p = 0.007), with higher levels at visit 2 and 8 than other visits. The dehydroepiandrosterone sulfate/dehydroepiandrosterone ratio significantly increased during treatment only in the verum group. Considering the adrenal zona glomerulosa, corticosterone significantly changed among visits both in both groups (p < 0.001), with higher levels at visit 2 (p = 0.028), 8 (p = 0.003), and 10 (p = 0.044), i.e., in coincidence with the complete clinical and instrumental examination performed only at these visits according to the study protocol. Chronically administered vardenafil reduces dehydroepiandrosterone levels and increases dehydroepiandrosterone sulfate/dehydroepiandrosterone ratio as possible consequences of modulation of steroidogenic enzymes by tissue changes in cyclic adenosine monophosphate and cyclic guanosine monophosphate availability. A possibly stress-related increase in corticosterone is suggested for the first time.

Does Aldosterone Play a Significant Role for Regulation of Vascular Tone?

Besides the well-known renal effects of aldosterone, the hormone is now known to have direct vascular effects. Clinical observations underline substantial adverse effects of aldosterone on cardiovascular function. The source of systemic circulating aldosterone is the adrenal gland zona glomerulosa cells through stimulus-secretion coupling involving depolarization, opening of L- and T-type calcium channels and aldosterone synthase activation. Local formation and release in peripheral tissues such as perivascular fat is recognized. Where does aldosterone affect the vasculature? Mineralocorticoid receptors (MRs) are present in endothelial and vascular smooth muscle cells, and MR-independent pathways are also involved. The vascular effects of aldosterone are complex, both concentration and temporal and spatial aspects are relevant. The acute response includes vasodilation through endothelial nitric oxide formation and vasoconstrictor effects through endothelial-contracting cyclooxygenase-derived factors and a changed calcium handling. The response to aldosterone can change within the same blood vessels depending on the exposure time and status of the endothelium. Chronic responses involve changed levels of reactive oxygen radicals, endothelial Na-influx and smooth muscle calcium channel expression. Furthermore, perivascular cells for example mast cells have also been suggested to participate in the chronic response. Moreover, the vascular effect of aldosterone depends on the status of the endothelium which is likely the cause of the very different responses to aldosterone and MR treatment observed in human studies going from increased to decreased flow depending on whether the patient had prior cardiovascular disease with endothelial dysfunction or not. A preponderance of constrictor versus dilator responses to aldosterone could therefore be involved in the detrimental vascular actions of the hormone in the setting of endothelial dysfunction and contribute to explain the beneficial action of MR blockers on blood pressure and target organ injury.

Role of ACTH and Other Hormones in the Regulation of Aldosterone Production in Primary Aldosteronism

The major physiological regulators of aldosterone production from the adrenal zona glomerulosa are potassium and angiotensin II; other acute regulators include adrenocorticotropic hormone (ACTH) and serotonin. Their interactions with G-protein coupled hormone receptors activate cAMP/PKA pathway thereby regulating intracellular calcium flux and CYP11B2 transcription, which is the specific steroidogenic enzyme of aldosterone synthesis. In primary aldosteronism (PA), the increased production of aldosterone and resultant relative hypervolemia inhibits the renin and angiotensin system; aldosterone secretion is mostly independent from the suppressed renin–angiotensin system, but is not autonomous, as it is regulated by a diversity of other ligands of various eutopic or ectopic receptors, in addition to activation of calcium flux resulting from mutations of various ion channels. Among the abnormalities in various hormone receptors, an overexpression of the melanocortin type 2 receptor (MC2R) could be responsible for aldosterone hypersecretion in aldosteronomas. An exaggerated increase in plasma aldosterone concentration (PAC) is found in patients with PA secondary either to unilateral aldosteronomas or bilateral adrenal hyperplasia (BAH) following acute ACTH administration compared to normal individuals. A diurnal increase in PAC in early morning and its suppression by dexamethasone confirms the increased role of endogenous ACTH as an important aldosterone secretagogue in PA. Screening using a combination of dexamethasone and fludrocortisone test reveals a higher prevalence of PA in hypertensive populations compared to the aldosterone to renin ratio. The variable level of MC2R overexpression in each aldosteronomas or in the adjacent zona glomerulosa hyperplasia may explain the inconsistent results of adrenal vein sampling between basal levels and post ACTH administration in the determination of source of aldosterone excess. In the rare cases of glucocorticoid remediable aldosteronism, a chimeric CYP11B2 becomes regulated by ACTH activating its chimeric CYP11B1 promoter of aldosterone synthase in bilateral adrenal fasciculate-like hyperplasia. This review will focus on the role of ACTH on excess aldosterone secretion in PA with particular focus on the aberrant expression of MC2R in comparison with other aberrant ligands and their GPCRs in this frequent pathology.

Immunohistochemistry of the adrenal in primary aldosteronism.

Primary aldosteronism is a major cause of hypertension; aldosterone-producing adenomas (APA) cause about half of primary aldosteronism; idiopathic hyperplasia of adrenal glomerulosa cells are responsible for the rest. A surprising variety of mutations have recently been identified in ion channels and pumps in a significant number of APA. The present review addresses histological and molecular aspects of APA and the surrounding adrenal. Specific antibodies against the CYP11B2 and CYP11B1 enzymes, the last enzyme in aldosterone and cortisol synthesis, respectively, allow for the first time study of the steroidogenic capabilities of cells within the APA and adjacent adrenal. Cells expressing CYP11B2 may be scattered and/or in clusters throughout the normal adrenal zona glomerulosa. APA differ widely in the number of cells expressing CYP11B2; some did not express it at all, but were surrounded by cells, some in clusters or micronodules, that expressed CYP11B2. Some APAs also comprised cells expressing both CYP11B1 and CYP17A1. In some samples, analysis of the tissue adjacent to APA detected ion channel and pump mutations heretofore associated only with APA. APAs have a complex structure and expression of steroidogenic enzymes.

Antiaging Gene Klotho Regulates Adrenal CYP11B2 Expression and Aldosterone Synthesis.

Deficiency of the antiaging gene Klotho (KL) induces renal damage and hypertension through unknown mechanisms. In this study, we assessed whether KL regulates expression of CYP11B2, a key rate-limiting enzyme in aldosterone synthesis, in adrenal glands. We found that haplodeficiency of KL(+/-) in mice increased the plasma level of aldosterone by 16 weeks of age, which coincided with spontaneous and persistent elevation of BP. Blockade of aldosterone actions by eplerenone reversed KL deficiency-induced hypertension and attenuated the kidney damage. Protein expression of CYP11B2 was upregulated in adrenal cortex of KL(+/-) mice. KL and CYP11B2 proteins colocalized in adrenal zona glomerulosa cells. Silencing of KL upregulated and overexpression of KL downregulated CYP11B2 expression in human adrenocortical cells. Notably, silencing of KL decreased expression of SF-1, a negative transcription factor of CYP11B2, but increased phosphorylation of ATF2, a positive transcription factor of CYP11B2, which may contribute to upregulation of CYP11B2 expression. Therefore, these results show that KL regulates adrenal CYP11B2 expression. KL deficiency-induced spontaneous hypertension and kidney damage may be partially attributed to the upregulation of CYP11B2 expression and aldosterone synthesis.

Adrenal histopathology in primary aldosteronism: is it time for a change?

Primary aldosteronism (PA) entails a group of disorders characterized by excess aldosterone production, relatively independent from the renin–angiotensin system.1 Because PA is the most common form of secondary hypertension and patients with PA have higher cardiovascular morbidity and mortality than age- and sex-matched patients with essential hypertension and the same degree of blood pressure elevation,2–5 early recognition of PA is of utmost importance to cure the high blood pressure and prevent deleterious cardiovascular effects. The 2 major PA subgroups are the unilateral forms, mainly aldosterone-producing adenoma (APA), and bilateral forms, mostly adrenal zona glomerulosa (ZG) hyperplasia, which require surgical or medical treatment, respectively. Adrenal vein sampling (AVS) is the best available method to distinguish between unilateral and bilateral aldosterone hypersecretion.6–8 In medical centers where AVS is available and strict cutoff values for the lateralization index are used,9 almost two thirds of PA cases are attributed to an APA, whereas one third remain classified as bilateral idiopathic forms. The opposite is seen when AVS is unavailable.10 Hence, the availability of AVS greatly affects the recognition of unilateral forms of PA that can be successfully treated with adrenalectomy. After surgery, demonstration of an adenoma at pathology is needed to reach a conclusive diagnosis of the only PA subtype that can be unequivocally diagnosed, eg, APA. After the introduction of the 4 corner criteria,10 an APA can be diagnosed when (1) there is evidence of PA at the screening tests, with an inappropriately high aldosterone/renin ratio and confirmation as needed; (2) aldosterone secretion is lateralized at AVS; (3) an adenoma is detected at pathology; and crucially important, (4) PA is corrected by adrenalectomy. However, at pathology, the identification of an APA may be challenging …

Expression of estrogen, estrogen related and androgen receptors in adrenal cortex of intact adult male and female rats.

Adrenocortical activity in various species is sensitive to androgens and estrogens. They may affect adrenal cortex growth and functioning either via central pathways (CRH and ACTH) or directly, via specific receptors expressed in the cortex and/or by interfering with adrenocortical enzymes, among them those involved in steroidogenesis. Only limited data on expression of androgen and estrogen receptors in adrenal glands are available. Therefore the present study aimed to characterize, at the level of mRNA, expression of these receptors in specific components of adrenal cortex of intact adult male and female rats. Studies were performed on adult male and female (estrus) Wistar rats. Total RNA was isolated from adrenal zona glomerulosa (ZG) and fasciculate/reticularis (ZF/R). Expression of genes were evaluated by means of Affymetrix® Rat Gene 1.1 ST Array Strip and QPCR. By means of Affymetrix® Rat Gene 1.1 ST Array we examined adrenocortical sex differences in the expression of nearly 30,000 genes. All data were analyzed in relation to the adrenals of the male rats. 32 genes were differentially expressed in ZG, and 233 genes in ZF/R. In the ZG expression levels of 24 genes were lower and 8 higher in female rats. The more distinct sex differences were observed in the ZF/R, in which expression levels of 146 genes were lower and 87 genes higher in female rats. Performed analyses did not reveal sex differences in the expression levels of both androgen (AR) and estrogen (ER) receptor genes in the adrenal cortex of male and female rats. Therefore matrix data were validated by QPCR. QPCR revealed higher expression levels of AR gene both in ZG and ZF/R of male than female rats. On the other hand, QPCR did not reveal sex-related differences in the expression levels of ERα, ERβ and non-genomic GPR30 (GPER-1) receptor. Of those genes expression levels of ERα genes were the highest. In studied adrenal samples the relative expression of ERα mRNA was higher than ERβ mRNA. In adrenals of adult male and female rats expression levels of estrogen-related receptors ERRα and ERRβ were similar, and only in the ZF/R of female rats ERRγ expression levels were significantly higher than in males. We also analyzed expression profile of three isoforms of steroid 5α-reductase (Srd5a1, Srd5a2 and Srd5a3) and aromatase (Cyp19a1) and expression levels of all these genes were similar in ZG and ZF/R of male and female rats. In contrast to Affymetrix microarray data QPCR revealed higher expression levels of AR gene in adrenal glands of the male rats. In adrenals of both sexes expression levels of ERa, ERb, non-genomic GPR30 (GPER-1), ERR α and ERRβ receptors were comparable. The obtained results suggest that acute steroidogenic effect of estrogens on corticosteroid secretion may be mediated by non-genomic GPR30.

Pendrin localizes to the adrenal medulla and modulates catecholamine release.

Pendrin (Slc26a4) is a Cl(-)/HCO3 (-) exchanger expressed in renal intercalated cells and mediates renal Cl(-) absorption. With pendrin gene ablation, blood pressure and vascular volume fall, which increases plasma renin concentration. However, serum aldosterone does not significantly increase in pendrin-null mice, suggesting that pendrin regulates adrenal zona glomerulosa aldosterone production. Therefore, we examined pendrin expression in the adrenal gland using PCR, immunoblots, and immunohistochemistry. Pendrin protein was detected in adrenal lysates from wild-type but not pendrin-null mice. However, immunohistochemistry and qPCR of microdissected adrenal zones showed that pendrin was expressed in the adrenal medulla, rather than in cortex. Within the adrenal medulla, pendrin localizes to both epinephrine- and norepinephrine-producing chromaffin cells. Therefore, we examined plasma catecholamine concentration and blood pressure in wild-type and pendrin-null mice under basal conditions and then after 5 and 20 min of immobilization stress. Under basal conditions, blood pressure was lower in the mutant than in the wild-type mice, although epinephrine and norepinephrine concentrations were similar. Catecholamine concentration and blood pressure increased markedly in both groups with stress. With 20 min of immobilization stress, epinephrine and norepinephrine concentrations increased more in pendrin-null than in wild-type mice, although stress produced a similar increase in blood pressure in both groups. We conclude that pendrin is expressed in the adrenal medulla, where it blunts stress-induced catecholamine release.

Morphometry in development of red deer’s adrenal glands

Histological structures and morphometric and some histochemical indicators of elk's adrenal gland development as subspecies of red deer in prenatal and postnatal ontogenies stages was studied. It was found that the growth of the fetus adrenal glands weight and the thickness of the structures adrenal glands fragments continue throughout the prenatal period of ontogeny. The cells of androgenic zone with single wandering sympathogoniae are differentiated in the adrenal glands in the second month of development. The androgenic and definite zone and the adrenal medulla are differentiated by the third month of development. At the 4 months, adrenal gland cortex zona glomerulosa and zona fasciculate-reticularis are differentiated; zona reticularis is differentiated only by the seventh month. By the eighth month, the structure of adrenal glands corresponds to the adrenal glands of a newborn. Full structural formation of the adrenal glands takes place in young animals by age 1.5. Obvious structural changes were not found late in the postnatal stages of development.

LGR5 Activates Noncanonical Wnt Signaling and Inhibits Aldosterone Production in the Human Adrenal.

Context:Aldosterone synthesis and cellularity in the human adrenal zona glomerulosa (ZG) is sparse and patchy, presumably due to salt excess. The frequency of somatic mutations causing aldosterone-producing adenomas (APAs) may be a consequence of protection from cell loss by constitutive aldosterone production.Objective:The objective of the study was to delineate a process in human ZG, which may regulate both aldosterone production and cell turnover.Design:This study included a comparison of 20 pairs of ZG and zona fasciculata transcriptomes from adrenals adjacent to an APA (n = 13) or a pheochromocytoma (n = 7).Interventions:Interventions included an overexpression of the top ZG gene (LGR5) or stimulation by its ligand (R-spondin-3).Main Outcome Measures:A transcriptome profile of ZG and zona fasciculata and aldosterone production, cell kinetic measurements, and Wnt signaling activity of LGR5 transfected or R-spondin-3-stimulated cells were measured.Results:LGR5 was the top gene up-regulated in ZG (25-fold). The gene for its cognate ligand R-spondin-3, RSPO3, was 5-fold up-regulated. In total, 18 genes associated with the Wnt pathway were greater than 2-fold up-regulated. ZG selectivity of LGR5, and its absence in most APAs, were confirmed by quantitative PCR and immunohistochemistry. Both R-spondin-3 stimulation and LGR5 transfection of human adrenal cells suppressed aldosterone production. There was reduced proliferation and increased apoptosis of transfected cells, and the noncanonical activator protein-1/Jun pathway was stimulated more than the canonical Wnt pathway (3-fold vs 1.3-fold). ZG of adrenal sections stained positive for apoptosis markers.Conclusion:LGR5 is the most selectively expressed gene in human ZG and reduces aldosterone production and cell number. Such conditions may favor cells whose somatic mutation reverses aldosterone inhibition and cell loss.

Bone Morphogenetic Protein-4 (BMP4): A Paracrine Regulator of Human Adrenal C19 Steroid Synthesis.

Bone morphogenetic proteins (BMPs) comprise one of the largest subgroups in the TGF-β ligand superfamily. We have identified a functional BMP system equipped with the ligand (BMP4), receptors (BMP type II receptor, BMP type IA receptor, also called ALK3) and the signaling proteins, namely the mothers against decapentaplegic homologs 1, 4, and 5 in the human adrenal gland and the human adrenocortical cell line H295R. Microarray, quantitative RT-PCR, and immunohistochemistry confirmed that BMP4 expression was highest in the adrenal zona glomerulosa followed by the zona fasciculata and zona reticularis. Treatment of H295R cells with BMP4 caused phosphorylation of the mothers against decapentaplegic and a profound decrease in synthesis of the C19 steroids dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione. Administration of BMP4 to cultures of H295R cells also caused a profound decrease in the mRNA and protein levels of 17α-hydroxylase/17,20-lyase (CYP17A1 and P450c17, respectively) but no significant effect on the mRNA levels of cholesterol side-chain cleavage cytochrome P450 (CYP11A1) or type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). Furthermore, Noggin (a BMP inhibitor) was able to reverse the negative effects of BMP4 with respect to both CYP17A1 transcription and DHEA secretion in the H295R cell line. Collectively the present data suggest that BMP4 is an autocrine/paracrine negative regulator of C19 steroid synthesis in the human adrenal and works by suppressing P450c17.

Role of Ca2+/Calmodulin-Dependent Protein Kinase Kinase in Adrenal Aldosterone Production

There is considerable evidence supporting the role of calcium signaling in adrenal regulation of both aldosterone synthase (CYP11B2) and aldosterone production. However, there have been no studies that investigated the role played by the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) in adrenal cells. In this study we investigated the role of CaMKK in adrenal cell aldosterone production. To determine the role of CaMKK, we used a selective CaMKK inhibitor (STO-609) in the HAC15 human adrenal cell line. Cells were treated with angiotensin II (Ang II) or K+ and evaluated for the expression of steroidogenic acute regulatory protein and CYP11B2 (mRNA/protein) as well as aldosterone production. We also transduced HAC15 cells with lentiviral short hairpin RNAs of CaMKK1 and CaMKK2 to determine which CaMKK plays a more important role in adrenal cell regulation of the calcium signaling cascade. The CaMKK inhibitor, STO-609, decreased aldosterone production in cells treated with Ang II or K+ in a dose-dependent manner. STO-609 (20 μM) also inhibited steroidogenic acute regulatory protein and CYP11B2 mRNA/protein induction. CaMKK2 knockdown cells showed significant reduction of CYP11B2 mRNA induction and aldosterone production in cells treated with Ang II, although there was no obvious effect in CaMKK1 knockdown cells. In immunohistochemical analysis, CaMKK2 protein was highly expressed in human adrenal zona glomerulosa with lower expression in the zona fasciculata. In conclusion, the present study suggests that CaMKK2 plays a pivotal role in the calcium signaling cascade regulating adrenal aldosterone production.

Sex-related gene expression profiles in the adrenal cortex in the mature rat: microarray analysis with emphasis on genes involved in steroidogenesis.

Notable sex-related differences exist in mammalian adrenal cortex structure and function. In adult rats, the adrenal weight and the average volume of zona fasciculata cells of females are larger and secrete greater amounts of corticosterone than those of males. The molecular bases of these sex-related differences are poorly understood. In this study, to explore the molecular background of these differences, we defined zone- and sex-specific transcripts in adult male and female (estrous cycle phase) rats. Twelve-week-old rats of both genders were used and samples were taken from the zona glomerulosa (ZG) and zona fasciculata/reticularis (ZF/R) zones. Transcriptome identification was carried out using the Affymetrix® Rat Gene 1.1 ST Array. The microarray data were compared by fold change with significance according to moderated t-statistics. Subsequently, we performed functional annotation clustering using the Gene Ontology (GO) and Database for Annotation, Visualization and Integrated Discovery (DAVID). In the first step, we explored differentially expressed transcripts in the adrenal ZG and ZF/R. The number of differentially expressed transcripts was notably higher in the female than in the male rats (702 vs. 571). The differentially expressed genes which were significantly enriched included genes involved in steroid hormone metabolism, and their expression levels in the ZF/R of adult female rats were significantly higher compared with those in the male rats. In the female ZF/R, when compared with that of the males, prevailing numbers of genes linked to cell fraction, oxidation/reduction processes, response to nutrients and to extracellular stimuli or steroid hormone stimuli were downregulated. The microarray data for key genes involved directly in steroidogenesis were confirmed by qPCR. Thus, when compared with that of the males, in the female ZF/R, higher expression levels of genes involved directly in steroid hormone synthesis were accompanied by lower expression levels of genes regulating basal cell functions.

Visinin-like peptide 1 in adrenal gland of the rat. Gene expression and its hormonal control

VSNL1 encodes the calcium-sensor protein visinin-like 1 and was identified previously as an upregulated gene in a sample set of aldosterone-producing adenomas. Recently, by means of microarray studies we demonstrated high expression of Vsnl1 gene in rat adrenal zona glomerulosa (ZG). Only scanty data are available on the role of this gene in adrenal function as well as on regulation of its expression by factors affecting adrenal cortex structure and function. Therefore we performed relevant studies aimed at clarifying some of the above issues. By Affymetrix® Rat Gene 1.1 ST Array Strip, QPCR and immunohistochemistry we demonstrated that expression levels of Vsnl1 in the rat adrenal ZG are notably higher than in the fasciculata/reticularis zone. In QPCR assay this difference was approximately 10 times higher. Expression of this gene in the rat adrenal gland or adrenocortical cells was acutely down regulated by ACTH, while chronic administration of corticotrophin or dexamethasone did not change Vsnl1 mRNA levels. In enucleation-induced adrenocortical regeneration expression levels of both Vsnl1 and Cyp11b2 were notably lowered and positively correlated. Despite these findings, the physiological significance of adrenal Vsnl1 remains unclear, and requires further investigation