Bioelectronic sensor mimicking the human neuroendocrine system for the detection of hypothalamic-pituitary-adrenal axis hormones in human blood.

In the neuroendocrine system, corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) play important roles in the regulation of the hypothalamic-pituitary-adrenal (HPA) system. Disorders of the HPA system lead to physiological problems, such as Addison's disease and Cushing's syndrome. Therefore, detection of CRH and ACTH is essential for diagnosing disorders related to the HPA system. Herein, receptors of the HPA axis were used to construct a bioelectronic sensor system for the detection of CRH and ACTH. The CRH receptor, corticotropin-releasing hormone receptor 1 (CRHR1), and the ACTH receptor, melanocortin 2 receptor (MC2R), were produced using an Escherichia coli expression system, and were reconstituted using nanodisc (ND) technology. The receptor-embedded NDs were immobilized on a floating electrode of a carbon nanotube field-effect transistor (CNT-FET). The constructed sensors sensitively detected CRH and ACTH to a concentration of 1 fM with high selectivity in real time. Furthermore, the reliable detection of CRH and ACTH in human plasma by the developed sensors demonstrated their potential in clinical and practical applications. These results indicate that CRHR1 and MC2R-based bioelectronic sensors can be applied for rapid and efficient detection of CRH and ACTH.

Molecular determinants of ACTH receptor for ligand selectivity.

The adrenocorticotropic hormone (ACTH) receptor, known as the melanocortin-2 receptor (MC2R), plays a key role in regulating adrenocortical function. ACTH receptor is a subtype of the melanocortin receptor family which is a member of the G-protein coupled receptor (GPCR) superfamily. ACTH receptor has unique characteristics among MCRs. α-MSH, ß-MSH, γ-MSH and ACTH are agonists for MCRs but only ACTH is the agonist for ACTH receptor. In addition, the melanocortin receptor accessory protein (MRAP) is required for ACTH receptor expression at cell surface and function. In this review, we summarized the information available on the relationship between ACTH and ACTH receptor and provide the latest understanding of the molecular basis of the ACTH receptor responsible for ligand selectivity and function.

Astrocytes Amplify Neuronal Dendritic Volume Transmission Stimulated by Norepinephrine.

In addition to their support role in neurotransmitter and ion buffering, astrocytes directly regulate neurotransmission at synapses via local bidirectional signaling with neurons. Here, we reveal a form of neuronal-astrocytic signaling that transmits retrograde dendritic signals to distal upstream neurons in order to activate recurrent synaptic circuits. Norepinephrine activates α1 adrenoreceptors in hypothalamic corticotropin-releasing hormone (CRH) neurons to stimulate dendritic release, which triggers an astrocytic calcium response and release of ATP; ATP stimulates action potentials in upstream glutamate and GABA neurons to activate recurrent excitatory and inhibitory synaptic circuits to the CRH neurons. Thus, norepinephrine activates a retrograde signaling mechanism in CRH neurons that engages astrocytes in order to extend dendritic volume transmission to reach distal presynaptic glutamate and GABA neurons, thereby amplifying volume transmission mediated by dendritic release.

Novel zebrafish behavioral assay to identify modifiers of the rapid, nongenomic stress response.

When vertebrates face acute stressors, their bodies rapidly undergo a repertoire of physiological and behavioral adaptations, which is termed the stress response. Rapid changes in heart rate and blood glucose levels occur via the interaction of glucocorticoids and their cognate receptors following hypothalamic-pituitary-adrenal axis activation. These physiological changes are observed within minutes of encountering a stressor and the rapid time domain rules out genomic responses that require gene expression changes. Although behavioral changes corresponding to physiological changes are commonly observed, it is not clearly understood to what extent hypothalamic-pituitary-adrenal axis activation dictates adaptive behavior. We hypothesized that rapid locomotor response to acute stressors in zebrafish requires hypothalamic-pituitary-interrenal (HPI) axis activation. In teleost fish, interrenal cells are functionally homologous to the adrenocortical layer. We derived eight frameshift mutants in genes involved in HPI axis function: two mutants in exon 2 of mc2r (adrenocorticotropic hormone receptor), five in exon 2 or 5 of nr3c1 (glucocorticoid receptor [GR]) and two in exon 2 of nr3c2 (mineralocorticoid receptor [MR]). Exposing larval zebrafish to mild environmental stressors, acute changes in salinity or light illumination, results in a rapid locomotor response. We show that this locomotor response requires a functioning HPI axis via the action of mc2r and the canonical GR encoded by nr3c1 gene, but not MR (nr3c2). Our rapid behavioral assay paradigm based on HPI axis biology can be used to screen for genetic and environmental modifiers of the hypothalamic-pituitary-adrenal axis and to investigate the effects of corticosteroids and their cognate receptor interactions on behavior.

Adrenocortical endocrine disruption.

The adrenal has been neglected in endocrine disruption regulatory testing strategy. The adrenal is a vital organ, adrenocortical insufficiency is recognised in life threatening "adrenal crises" and Addison's disease, and the consequences of off-target toxicological inhibition of adrenocortical steroidogenesis is well recognised in clinical medicine, where drugs such as aminoglutethimide and etomidate killed patients via unrecognised inhibition of adrenocortical steroidogenic enzymes (e.g. CYP11B1) along the cortisol and aldosterone pathways. The consequences of adrenocortical dysfunction during early development are also recognised in the congenital salt wasting and adrenogenital syndromes presenting neonatally, yet despite a remit to focus on developmental and reproductive toxicity mechanisms of endocrine disruption by many regulatory agencies (USEPA EDSTAC; REACH) the assessment of adrenocortical function has largely been ignored. Further, every step in the adrenocortical steroidogenic pathway (ACTH receptor, StAR, CYP's 11A1, 17, 21, 11B1, 11B2, and 3-hydroxysteroid dehydrogenase Δ4,5 isomerase) is known to be a potential target with multiple examples of chemicals inhibiting these targets. Many of these chemicals have been detected in human and wildlife tissues. This raises the question of whether exposure to low level environmental chemicals may be affecting adrenocortical function. This review examines the omission of adrenocortical testing in the current regulatory frameworks; the characteristics that make the adrenal cortex particularly vulnerable to toxic insult; chemicals and their toxicological targets within the adrenocortical steroidogenic pathways; the typical manifestations of adrenocortical toxicity (e.g. human iatrogenically induced pharmacotoxicological adrenal insufficiency, manifestations in typical mammalian regulatory general toxicology studies, manifestations in wildlife) and models of adrenocortical functional assessment. The utility of the in vivo ACTH challenge test to prove adrenocortical competency, and the H295R cell line to examine molecular mechanisms of steroidogenic pathway toxicity, are discussed. Finally, because of the central role of the adrenal in the physiologically adaptive stress response, the distinguishing features of stress, compared with adrenocortical toxicity, are discussed with reference to the evidence required to claim that adrenal hypertrophy results from stress rather than adrenocortical enzyme inhibition which is a serious adverse toxicological finding. This article is part of a special issue entitled 'Endocrine disruptors and steroids'.

Third transmembrane domain of the adrenocorticotropic receptor is critical for ligand selectivity and potency.

The ACTH receptor, known as the melanocortin-2 receptor (MC2R), plays an important role in regulating and maintaining adrenocortical function. MC2R is a subtype of the melanocortin receptor (MCR) family and has unique characteristics among MCRs. Endogenous ACTH is the only endogenous agonist for MC2R, whereas the melanocortin peptides α-, ß-, and γ-melanocyte-stimulating hormone and ACTH are full agonists for all other MCRs. In this study, we examined the molecular basis of MC2R responsible for ligand selectivity using ACTH analogs and MC2R mutagenesis. Our results indicate that substitution of Phe(7) with D-Phe or D-naphthylalanine (D-Nal(2')) in ACTH(1-24) caused a significant decrease in ligand binding affinity and potency. Substitution of Phe(7) with D-Nal(2') in ACTH(1-24) did not switch the ligand from agonist to antagonist at MC2R, which was observed in MC3R and MC4R. Substitution of Phe(7) with D-Phe(7) in ACTH(1-17) resulted in the loss of ligand binding and activity. Molecular analysis of MC2R indicated that only mutation of the third transmembrane domain of MC2R resulted in a decrease in D-Phe ACTH binding affinity and potency. Our results suggest that Phe(7) in ACTH plays an important role in ligand selectivity and that the third transmembrane domain of MC2R is crucial for ACTH selectivity and potency.

Dynamic pituitary-adrenal interactions in response to cardiac surgery.

OBJECTIVES: To characterize the dynamics of the pituitary-adrenal interaction during the course of coronary artery bypass grafting both on and off pump. Since our data pointed to a major change in adrenal responsiveness to adrenocorticotropic hormone, we used a reverse translation approach to investigate the molecular mechanisms underlying this change in a rat model of critical illness. CLINICAL STUDIES: Prospective observational study. ANIMAL STUDIES: Controlled experimental study. CLINICAL STUDIES: Cardiac surgery operating rooms and critical care units. ANIMAL STUDIES: University research laboratory. CLINICAL STUDIES: Twenty, male patients. ANIMAL STUDIES: Adult, male Sprague-Dawley rats. CLINICAL STUDIES: Coronary artery bypass graft-both on and off pump. ANIMAL STUDIES: Injection of either lipopolysaccharide or saline (controls) via a jugular vein cannula. CLINICAL STUDIES: Blood samples were taken for 24 hours from placement of the first venous access. Cortisol and adrenocorticotropic hormone were measured every 10 and 60 minutes, respectively, and corticosteroid-binding globulin was measured at the beginning and end of the 24-hour period and at the end of operation. There was an initial rise in both levels of adrenocorticotropic hormone and cortisol to supranormal values at around the end of surgery. Adrenocorticotropic hormone levels then returned toward preoperative values. Ultradian pulsatility of both adrenocorticotropic hormone and cortisol was maintained throughout the perioperative period in all individuals. The sensitivity of the adrenal gland to adrenocorticotropic hormone increased markedly at around 8 hours after surgery maintaining very high levels of cortisol in the face of "basal" levels of adrenocorticotropic hormone. This sensitivity began to return toward preoperative values at the end of the 24-hour sampling period. ANIMAL STUDIES: Adult, male Sprague-Dawley rats were given either lipopolysaccharide or sterile saline via a jugular vein cannula. Hourly blood samples were subsequently collected for adrenocorticotropic hormone and corticosterone measurement. Rats were killed 6 hours after the injection, and the adrenal glands were collected for measurement of steroidogenic acute regulatory protein, steroidogenic factor 1, and dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 messenger RNAs and protein using real-time quantitative polymerase chain reaction and Western immunoblotting, respectively. Adrenal levels of the adrenocorticotropic hormone receptor (melanocortin type 2 receptor) messenger RNA and its accessory protein (melanocortin type 2 receptor accessory protein) were also measured by real-time quantitative polymerase chain reaction. In response to lipopolysaccharide, rats showed a pattern of adrenocorticotropic hormone and corticosterone that was similar to patients undergoing coronary artery bypass grafting. We were also able to demonstrate increased intra-adrenal corticosterone levels and an increase in steroidogenic acute regulatory protein, steroidogenic factor 1, and melanocortin type 2 receptor accessory protein messenger RNAs and steroidogenic acute regulatory protein, and a reduction in dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 and melanocortin type 2 receptor messenger RNAs, 6 hours after lipopolysaccharide injection. CONCLUSIONS: Severe inflammatory stimuli activate the hypothalamic-pituitary-adrenal axis resulting in increased steroidogenic activity in the adrenal cortex and an elevation of cortisol levels in the blood. Following coronary artery bypass grafting, there is a massive increase in both adrenocorticotropic hormone and cortisol secretion. Despite a subsequent fall of adrenocorticotropic hormone to basal levels, cortisol remains elevated and coordinated adrenocorticotropic hormone-cortisol pulsatility is maintained. This suggested that there is an increase in adrenal sensitivity to adrenocorticotropic hormone, which we confirmed in our animal model of immune activation of the hypothalamic-pituitary-adrenal axis. Using this model, we were able to show that this increased adrenal sensitivity results from changes in the regulation of both stimulatory and inhibitory intra-adrenal signaling pathways. Increased understanding of the dynamics of normal hypothalamic-pituitary-adrenal responses to major surgery will provide us with a more rational approach to glucocorticoid therapy in critically ill patients.

How does stress possibly affect cardiac remodeling?

The aim of this study was to evaluate the predictive value of adrenocorticotropic hormone (ACTH), cortisol and ACTH receptor polymorphism (ACTHRP) for left ventricular (LV) remodeling. Thirty-six elite male athletes, as chronic stress adaptation models, and twenty sedentary age and sex-mached subjects emabarked on standard and tissue Doppler echocardiography to assess cardiac parameters at rest. They performed maximal cardiopulmonary test, which was used as an acute stress model. ACTH and cortisol were measured at rest (10min before test), at beginning, at maximal effort, at 3rd min of recovery, using radioimmunometric and radioimmunoassey techniques, respectively. Promoter region of ACTHR gene (18p11.2) was analysed from blood samples using reverse polymerization reaction with the analysis of restriction fragment length polimorphisam by SacI restriction enzyme. Normal genotype was CTC/CTC, heterozygot for ACTHRP CTC/CCC and homozygot CCC/CCC. In all participants, ACTH and cortisol increased during acute stress, whereas in recovery ACTH increased and cortisol remained unchanged. 49/56 examiners manifested CTC/CTC, 7/56 CTC/CCC and 0/56 CCC/CCC. There was no difference in ACTHRP frequency between groups (χ(1)(2)=0.178, p=0.67). LV mass (LVM) and LV end-diastolic volume (LVVd) were higher in athletes than in controls (p<0.01) and lower in CTC/CTC than in CTC/CCC genotype (219.43±46.59(SD)g vs. 276.34±48.86(SD)g, p=0.004; 141.24±24.46(SD)ml vs. 175.29±37.07(SD)ml, p=0.002; respectively). In all participants, predictors of LVM and LVVd were ACTH at rest (B=-1.00,-0.44; ß=-0.30,-0.31; p=0.026,0.012, respectively) and ACTHRP (B=56.63,34; ß=0.37,0.40; p=0.003,0.001, respectively). These results demonstrate that ACTH and ACTHRP strongly predict cardiac morphology suggesting possible regulatory role of stress system activity and sensitivity in cardiac remodeling.

Long-term dysregulation of brain corticotrophin and glucocorticoid receptors and stress reactivity by single early-life pain experience in male and female rats.

Inflammatory pain experienced on the day of birth (postnatal day 0: PD0) significantly dampens behavioral responses to stress- and anxiety-provoking stimuli in adult rats. However, to date, the mechanisms by which early life pain permanently alters adult stress responses remain unknown. The present studies examined the impact of inflammatory pain, experienced on the day of birth, on adult expression of receptors or proteins implicated in the activation and termination of the stress response, including corticotrophin releasing factor receptors (CRFR1 and CRFR2) and glucocorticoid receptor (GR). Using competitive receptor autoradiography, we show that Sprague Dawley male and female rat pups administered 1% carrageenan into the intraplantar surface of the hindpaw on the day of birth have significantly decreased CRFR1 binding in the basolateral amygdala and midbrain periaqueductal gray in adulthood. In contrast, CRFR2 binding, which is associated with stress termination, was significantly increased in the lateral septum and cortical amygdala. GR expression, measured with in situ hybridization and immunohistochemistry, was significantly increased in the paraventricular nucleus of the hypothalamus and significantly decreased in the hippocampus of neonatally injured adults. In parallel, acute stress-induced corticosterone release was significantly attenuated and returned to baseline more rapidly in adults injured on PD0 in comparison to controls. Collectively, these data show that early life pain alters neural circuits that regulate responses to and neuroendocrine recovery from stress, and suggest that pain experienced by infants in the Neonatal Intensive Care Unit may permanently alter future responses to anxiety- and stress-provoking stimuli.

Melanocortin 4 receptor becomes an ACTH receptor by coexpression of melanocortin receptor accessory protein 2.

Melanocortin 2 receptor (MC2R) is the only canonical ACTH receptor. Its functional expression requires the presence of an accessory protein, known as melanocortin receptor 2 accessory protein 1 (MRAP1). The vertebrate genome exhibits a paralogue gene called MRAP2, which is duplicated in zebrafish (MRAP2a and MRAP2b), although its function remains unknown. In this paper, we demonstrate that MRAP2a enables MC4R, a canonical MSH receptor, to be activated by ACTH with a similar sensitivity to that exhibited by MC2R. Both proteins physically interact and are coexpressed in the neurons of the preoptic area, a key region in the control of the energy balance and hypophyseal secretion in fish. ACTH injections inhibit food intake in wild-type zebrafish but not in fish lacking functional MC4R. Both MRAP1 and MRAP2a are hormonally regulated, suggesting that these proteins are substrates for feed-back regulatory pathways of melanocortin signaling. Fasting has no effect on the central expression of MRAP2a but stimulates MRAP2b expression. This protein interacts and is colocalized with MC4R in the tuberal hypothalamic neurons but has no effect on the pharmacologic profile of MC4R. However, MRPA2b is able to decrease basal reporter activity in cell lines expressing MC4R. It is plausible that MRAP2b decreases the constitutive activity of the MC4R during fasting periods, driving the animal toward a positive energy balance. Our data indicate that MRAP2s control the activity of MC4R, opening up new pathways for the regulation of melanocortin signaling and, by extension, for the regulation of the energy balance and obesity.

Antenatal glucocorticoid exposure enhances the inhibition of adrenal steroidogenesis by leptin in a sex-specific fashion.

Antenatal treatment with glucocorticoids (GC) poses long-lasting effects on endocrine and cardiovascular function. Given that leptin attenuates adrenal function and the reported sex differences in plasma leptin concentration, we hypothesized that antenatal GC will affect leptin levels and leptin modulation of adrenal function in a sex-specific manner. Pregnant sheep were randomly given betamethasone or vehicle at 80 days of gestational age, and offspring were allowed to deliver at term. Adrenocortical cells (ADC) were studied from male and female animals at 1.5 yr of age. Plasma leptin was increased 66% in male and 41% in female GC-treated animals (P < 0.05), but adrenal leptin mRNA was increased only in GC-treated males (P < 0.05). Whereas mRNA expression of adrenal leptin receptor isoforms showed sex (Ob-Ra and Ob-Rb) and treatment-dependent (Ob-Rb) differences, protein expression remained unchanged. GC-treated females showed greater plasma cortisol and greater ACTH-stimulated cortisol production (P < 0.05) in ADC. Leptin exerted a greater inhibitory effect on basal and stimulated cortisol by ADC from GC-treated males (P < 0.05), with no differences in females. Similarly, greater inhibitory effects on basal and ACTH-stimulated StAR and ACTH-R mRNA expression by leptin were observed in cells from GC males (P < 0.05), with no changes in females. Persistent effects of antenatal GC on leptin levels and leptin modulation of adrenal function are expressed in a sex-specific manner; males are more sensitive than females to the inhibitory influences of leptin on adrenal function, and this effect appears to be mediated by a greater inhibition of StAR and ACTH-R expression in adrenals of adult GC-treated males.

ACTH resistance: genes and mechanisms.

ACTH resistance is a rare disorder typified by familial glucocorticoid deficiency (FGD), a genetically heterogeneous disease. Previously, genetic defects in FGD have been identified in the ACTH receptor gene (MC2R), its accessory protein (MRAP) and the steroidogenic acute regulatory protein gene (STAR). The defective mechanisms here are failures in ACTH ligand binding and/or receptor trafficking for MC2R and MRAP and, in the case of STAR mutations, inefficient cholesterol transport to allow steroidogenesis to proceed. Novel gene defects in FGD have recently been recognised in mini-chromosome maintenance-deficient 4 homologue (MCM4) and nicotinamide nucleotide transhydrogenase (NNT). MCM4 is one part of a DNA repair complex essential for DNA replication and genome stability, whilst NNT is involved in the glutathione redox system that protects cells against reactive oxygen species. The finding of mutations in these two genes implicates new pathogenetic mechanisms at play in FGD, and implies that the adrenal cortex is exquisitely sensitive to replicative and oxidative stresses.

Glucocorticoid receptor and molecular chaperones in the pathogenesis of adrenal incidentalomas: potential role of reduced sensitivity to glucocorticoids.

Glucocorticoid (GC) sensitivity depends on glucocorticoid receptor (GR) and heat shock proteins (Hsps). We investigated whether common GR genes (ER22/23EK, N363S, Bcl I, and 9ß) and adrenocorticotropin receptor promoter polymorphisms influence susceptibility for unilateral adrenal incidentaloma (AI), plus GR and Hsp expression in tumorous (n = 19), peritumorous (n = 13) and normal adrenocortical (n = 11) tissues. Patients (n = 112), population-matched controls (n = 100) and tumor tissues (n = 32) were genotyped for these polymorphisms. Postdexamethasone serum cortisol was higher in patients (p < 0.001). GR gene variants, larger allele of Bcl I (odds ratio [OR] 2.9; 95% confidence interval [CI] 1.7-5.1; p < 0.001] and minor allele of 9ß (OR 3.0; 95% CI 1.6-5.7; p < 0.001) were independent predictors of AI. In patients, the first allele is linked with larger tumors (p = 0.002) and the latter with higher postdexamethasone cortisol levels (p = 0.025). Both allele carriers had lesser waist circumference (p = 0.02), similar adrenocorticotropin and higher basal (p = 0.024) and postdexamethasone cortisol concentrations (p < 0.001). Tumorous and constitutional genotypes were similar. GR-D is the major receptor isoform in normal adrenal cortex by Western blotting. Loss of other receptor isoforms, decrease in immunostaining for GR (p < 0.0001), underexpression of chaperones (p ≤ 0.01) and the presence of inducible Hsp70 were found in adenomas. In conclusion, GR gene variants, C allele of Bcl I and minor allele of 9ß, are associated with AIs. Their concurrent presence in patients reduces GC sensitivity. Normal adrenal cortex preferentially expresses GR-D. In adenomas, the lack of other GR isoforms and underexpression of heat shock proteins perhaps permanently impair GC signaling, which could promote dysregulated cortisol production and tumor growth. The innate GC sensitivity probably modifies these effects.

Control of CYP11B2/CYP11B1 expression ratio and consequences for the zonation of the adrenal cortex.

Access of corticotropin to glucocorticoid synthesis in adrenocortical cells is provided by the expression of the ACTH receptor (MC2R). Activation of the MC2R increases stimulatory G-protein, adenylyl cyclase, and protein kinase A (PKA) activities. Furthermore, PKA phosphorylates transcription factors that have a stimulating effect on glucocorticoid synthesis. Sensitivity of adrenocortical cells to renin/angiotensin-2 is conferred by the expression of the inhibitory G-protein-linked angiotensin-2 type 1 receptor (AT1R) that additionally associates to the phospholipase C-activating G-protein q. The AT1R is connected to the adrenal potassium sensory system and regulates calcium influx as well as phospholipase C-ß (PLC-ß) and thus calmodulin kinase-dependent transcription of steroidogenic enzymes. While AT1R signaling suppresses the influence of corticotropin on the generation of cyclic adenosine monophosphate, the expression of the AT1R and its associated enzyme activities are under the control of glucocorticoids. Thus, dominance of one of the two signaling pathways is dependent on two factors: the extracellular concentration of their ligands and the products of their signaling pathways. These findings are in favor of the hypothesis that the centripetal blood flow through the adrenal gland builds up a glucocorticoid gradient creating a morphogenetic field along which adrenal cortical cells adopt different functional states, leading to the typical zonation of the adrenal cortex.

The -2T/C polymorphism in the adrenocorticotropin receptor gene affects stress perception of patients with alopecia areata.

BACKGROUND: Altered hypothalamic-pituitary-adrenal (HPA) axis response involved in the pathogenesis of stress-associated alopecia areata (AA) has been reported. A novel polymorphism -2T>C of the adrenocorticotropin receptor (ACTHR) can result in an insufficient HPA response to stress; therefore, the functional polymorphism may underlie a role in stress-associated AA. OBJECTIVE: To investigate the relationship between psychosocial factors and the risk of developing AA and to detect the association between the -2T>C polymorphism of ACTHR and AA. METHODS: Stressful situations were evaluated using Holmes and Rahe's social readjustment rating scale. The ACTHR -2T>C polymorphism was examined in 263 patients with AA and 241 controls. RESULTS: Significant elevation of psychological stress experienced by some patients with AA compared with controls (Z = 6.628, P < 0.01). The frequency of the ACTHR C allele showed a significant difference between patients with AA and controls (P = 0.004). Allele C is the risk allele with a dominant model as the -2C allele occurred more often in patients with AA (P = 0.001). There were significant differences between patients with AA with a severe stress attack versus patients with AA with no obvious stress (P < 0.001), whereas the genotype frequencies were not correlated with the type, duration of disease, and age at onset. Notably, the C allele carrier was significantly associated with stress risk in both AA and controls (P = 0.002, OR = 1.576, 95% CI: 1.148-2.162; P = 0.042, OR = 1.529, 95% CI: 1.022-2.288). CONCLUSIONS: These findings suggest AA in some patients may be associated with stress. The ACTHR gene -2T>C variant may be one important factor that influences stress perception of patients with AA.

Expression of aldosterone synthase and adrenocorticotropic hormone receptor in adrenal incidentalomas from normotensive and hypertensive patients: Distinguishing subclinical or atypical primary aldosteronism from adrenal incidentaloma.

The present study aimed to investigate the expression of aldosterone synthase (CYP11B2), adrenocorticotropic hormone receptor (ACTH-R) and their regulating transcription factors in adrenal incidentalomas (AIs) from normotensive and hypertensive patients to distinguish subclinical or atypical primary aldosteronism (PA) from AIs. Total RNA was extracted from 8 normal adrenal cortices (NAs), 46 AIs, 15 aldosterone-producing adenomas (APAs) and 6 idiopathic hyperaldosteronisms (IHAs). Real-time quantitative polymerase chain reaction (PCR) and immunohistochemistry were performed to determine the mRNA and protein expression of CYP11B2, ACTH-R, steroidogenic factor-1 (SF1) and dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1 (DAX-1) in the different tissues. The AI hypertensive subgroup displayed increased plasma aldosterone concentration (PAC) and PAC/PRA ratio (ARR) and decreased plasma renin activity (PRA) compared to the normotensive group. CYP11B2, ACTH-R and SF1 mRNAs were significantly higher in the APA group compared to the other groups, and gradually increased in AI hypertensive samples. DAX-1 mRNA was expressed faintly in PA compared with NA. In normotensive-AI samples, DAX-1 mRNA was higher compared to PA and AI hypertensive samples. Significant differences in gene expression levels in AIs were observed between probable and improbable PA patients. Immunohistochemical results were consistent with those of real-time PCR. Plasma aldosterone levels were positively correlated with CYP11B2, ACTH-R and SF1 mRNA and inversely correlated with DAX-1 mRNA. In conclusion, a significant number of hypertensive-AI patients may have subclinical forms of PA. CYP11B2, ACTH-R and their regulating transcription factors may be noteworthy in distinguishing subclinical PA from AIs.

Familial glucocorticoid deficiency in five Arab kindreds with homozygous point mutations of the ACTH receptor (MC2R): genotype and phenotype correlations.

BACKGROUND/AIMS: Familial glucocorticoid deficiency type 1 (FGD1) is a rare autosomal-recessive disorder resulting from defective ACTH receptor (melanocortin receptor type 2, MC2R). Individuals with this condition usually present in infancy or early childhood with the signs and symptoms of isolated glucocorticoid deficiency. To date, hypothyroidism has been reported as an associated feature in a few cases. The clinical findings along with MC2R genetic analysis of five Arab kindreds are described. SUBJECTS/METHODS: The subjects were children with the clinical and biochemical features of FGD1. Three patients had associated thyroid dysfunction and two patients had associated growth hormone deficiency (GHD). Mutation analysis of MC2R was performed by direct gene sequencing. RESULTS: Analysis of the MC2R gene revealed a homozygous insertion of a cytosine nucleotide between codons 153 and 154 (c.459_460insC) in all of the patients. This mutation would be expected to cause a translation frame shift after codon 154 and a premature termination codon at 248 of the MC2R mRNA (p.I154fsX248). CONCLUSIONS: Associated thyroid dysfunction and GHD were clinical features in the Bedouin patients with FGD1 caused by identical homozygous frameshift mutation in the MC2R gene.

[Associations of GR and ACTHR gene polymorphisms with quantitative trait of strain].

OBJECTIVE: To explore association between GR and ACTHR gene polymorphisms and quantitative trait of stress in Chinese Han population. METHODS: Four polymorphic markers (GRA5556G, A5556G, GAGG4534/4536AAAG, promoter T-2C) in GR gene and ACTHR gene were genotyped with PCR-RLFP in 200 healthy Hans. ISTA6.0 and life event stressor questionnaire was used to assess stressors. JSS, SCL-90 and GWB questionnaires were used to quantify the phenotypes of stress. Blood cortical and ACTH levels, and nervous behavior function were measured to assess Physiological strain. CWAI questionnaire was used to assess work ability. Then strain was assessed with Structural equation modeling (SEM). RESULTS: The subjects with GR A5556G genotype (G/A) showed significantly higher plasma cortisol levels, higher psychological stress scores, lower work ability scores and lower plasma ACTH levels compared with the subjects with wild-type (P < 0.01). Psychological stress scores and plasma cortisol levels in the subjects with GR GAGG4534/4536AAAG AG genotype were significantly higher than those in the subjects with wild-type, but the reaction and action sensitivity in the subjects with GR GAGG4534/4536AAAG AG genotype were significantly lower than those in ones with wild-type (P < 0.01). The ACTH level in the subjects with ACTHR promoter T-2C T/T genotype was significantly lower than that in ones with C/C and C/T genotype (P < 0.01). Interaction of GRA5556G and GG4534/4536AAAG with plasma cortisol was positively associated (ßs = 0.543, P < 0.01), but with SCL-90 score was negatively associated (ßs = -0.374, P < 0.01). Interaction of GRA5556G and GGC6294G with plasma cortisol was correlated (ßs = 0.465, P < 0.05). While GR and ACTHR gene variants are the risk factors for psychological strain, physiological strain and decreased work ability (ßs are 0.62, 0.43, -0.74, respectively (P < 0.01). While scarce social support, job stressors, negative life stressors and dangerous individual characters are the risk factors for occupational strain, psychological strain, physiological strain and decreased work ability (P < 0.01). CONCLUSION: GRA5556G, GRA5556G, GAGG4534/4536AAAG and ACTHR promoter T-2C variants might be associated with quantitative trait of strain, and GR and ACTHR gene variants with stressors increased the risk for developing strain.

Expression of the ACTH receptor, steroidogenic acute regulatory protein, and steroidogenic enzymes in canine cortisol-secreting adrenocortical tumors.

Studies of human adrenocortical tumors (ATs) causing Cushing's syndrome suggest that hypersecretion of cortisol is caused by altered expression of steroidogenic enzymes and that steroidogenesis can only be maintained when there is expression of the ACTH receptor (ACTH-R). Here we report the screening for the mRNA expression of the ACTH-R, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase, 21-hydroxylase (all in 38 cortisol-secreting ATs), 17α-hydroxylase, and 11ß-hydroxylase (both in 28 cortisol-secreting ATs). Real-time PCR (RT-PCR) was applied in all samples and was compared with that in normal canine adrenal glands. Messenger-RNA encoding StAR, steroidogenic enzymes, and ACTH-R were present in both normal adrenal glands and cortisol-secreting ATs. The amounts of mRNA encoding StAR and enzymes of the steroidogenic cluster needed for cortisol production did not differ significantly between either adenomas or carcinomas and normal adrenal glands. The amount of mRNA encoding ACTH-R was significantly lower in carcinomas than in normal adrenal glands (P = 0.008). In conclusion, RT-PCR analysis revealed no overexpression of StAR and steroidogenic enzymes in canine cortisol-secreting ATs. Significant downregulation of ACTH-R in carcinomas might be associated with the malignant character of the AT.

Aberrant hormone receptors in primary aldosteronism.

The mechanisms involved in the renin-independent regulation of aldosterone secretion in primary aldosteronism are poorly understood. In ACTH-independent Cushing's syndrome, cortisol secretion can be regulated by the aberrant expression of G-protein coupled receptors (GPCRs) in unilateral tumors and bilateral macronodular adrenal hyperplasia. By analogy, some recent studies identified overexpression or function of several GPCR as a potential cause for excess aldosterone production in some aldosteronomas and in bilateral idiopathic hyperaldosteronism. Initial studies have used in vitro techniques, while the clinical aldosterone responses were not assessed. More recently, several receptors have been shown to be expressed in aldosterone-producing adrenal tumors in vitro and to regulate aberrantly renin-independent aldosterone secretion in vivo. The prevalence of aberrant hormone receptors in primary aldosteronism could be elevated, but larger systematic studies are required to establish its true frequency. The identification of aberrant adrenal GPCRs by in vivo functional studies offers the potential for novel pharmacological therapies that either suppress the endogenous ligands or block the receptor with specific antagonists.