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The adrenocorticotropic hormone (ACTH) receptor, known as the melanocortin-2 receptor (MC2R), plays a key role in regulating adrenocortical function. MC2R is a subtype of the melanocortin receptor family and ACTH is only agonist for MC2R. Our previous result indicates that ACTH1-17 is the minimal peptide required for MC2R activation but DPhe7-ACTH1-17 has no activity at MC2R. In this study, we examined the molecular basis of the MC2R responsible for ligand selectivity using ACTH analogues and MC2R mutagenesis. Our results indicate that substitution of the 3TM of the MC2R with the corresponding region of the MC3R switches DPhe-ACTH1-17 from no activity to agonist. Further experiment indicates that substitution of the amino acid residue leucine to isoleucine in 112 (L112I) of the 3TM of the MC2R changes both DPhe-ACTH1-17 and ACTH1-15 from no activity to agonists. Surprisingly, mutation L112I switches α-MSH from no activity to agonist, suggesting that this residue plays a key role at MC2R for ligand ACTH or α-MSH selectivity.

Tritium-labeled synthetic fragments of human adrenocorticotropic hormone (ACTH) [3H]ACTH (11–24) and [3H]ACTH (15–18) with a specific activity of 22 and 26 Ci/mmol, respectively, were obtained. It was found that [3H]ACTH-(11–24) binds to membranes of the rat adrenal cortex with high affinity and high specificity (K d 1.8 ± 0.1 nM). Twenty nine fragments of ACTH (11–24) were synthesized, and their ability to inhibit the specific binding of [3H]ACTH (11–24) to adrenocortical membranes was investigated. The shortest active peptide was found to be an ACTH fragment (15–18) (KKRR) (K i 2.3 ± 0.2 nM), whose [3H] labeled derivative binds to rat adrenocortical membranes (K d 2.1 ± 0.1 nM) with a high affinity. The specific binding of [3H]ACTH-(15–18) was inhibited by 100% by unlabeled ACTH (11–24) (K i 2.0 ± 0.1 nM). ACTH (15–18) in the concentration range of 1–1000 nM did not affect the adenylate cyclase activity of adrenocortical membranes and, therefore, is an antagonist of the ACTH receptor.

The late-gestation plasma cortisol surge in the sheep fetus is critical for stimulating organ development and parturition. Increased adrenal responsiveness is one of the key reasons for the surge; however, the underlying mechanisms are not fully understood. Our recent studies suggest that ACTH-mediated increased expression of ACTH receptor (ACTH-R) and steroid acute regulatory protein (StAR) may play a role in enhancing responsiveness. Hence, we examined effects of ACTH infusion in fetal sheep on mRNA expression of these two mediators of adrenal responsiveness and assessed the functional consequences of this treatment in vitro. Fetuses of ∼118 and 138 days of gestational age (dGA) were infused with ACTH-(1–24) for 24 h. Controls received saline infusion. Arterial blood was sampled throughout the infusion. Adrenals were isolated and analyzed for ACTH-R and StAR mRNA, or cells were cultured for 48 h. Cells were stimulated with ACTH, and medium was collected for cortisol measurement. Fetal plasma ACTH and cortisol concentrations increased over the infusion period in both groups. ACTH-R mRNA levels were significantly higher in ACTH-infused fetuses in both the 118 and 138 dGA groups. StAR mRNA increased significantly in both the 118 and 138 dGA groups. Adrenal cells from ACTH-infused fetuses were significantly more responsive to ACTH stimulation in terms of cortisol secretion than those from saline-infused controls. These findings demonstrate that increases in circulating ACTH levels promote increased expression of ACTH-R and StAR mRNA and are coupled to heightened adrenal responsiveness.

Preparing the mammalian fetus for birth requires an increase in fetal plasma glucocorticoid levels. The mechanisms facilitating this increase are not fully known. It has been shown in sheep that the prepartum elevation in fetal plasma cortisol is accompanied by increases in adrenocorticotropin receptor (ACTH-R) expression in the fetal adrenal and in the adrenal responsiveness to stimulation. To determine the significance of the upregulation in ACTH-R expression on fetal adrenal function, the authors used small interfering RNA targeted to the ovine ACTH-R to reduce receptor expression and studied responses to stimulation in ovine adrenal cells. They studied fetal cells from late gestation after responsiveness had increased. They also studied adult cells to determine if maturation would influence the impact of receptor expression suppression on responsiveness. Fetal and adult cells were obtained, dispersed, transfected with receptor-targeted small interfering RNA or scrambled small interfering RNA, and subsequently stimulated with ACTH. Cells and media were harvested for measurements of gene and protein expression and cyclic adenosine monophosphate (cAMP) and cortisol levels. The ability of ACTH to upregulate its receptor or steroid acute regulatory protein was attenuated in fetal (P < .01) and adult cells (P < .01) by small interfering RNA treatment; the blockade was more pronounced in the adult cells (P < .01). The small interfering RNA treatment also blocked the cAMP response to ACTH in fetal (P < .001) and adult (P < .05) cells. This was accompanied by marked reductions in cortisol responses in both (P < .001 and P < .01, respectively). These data suggest that upregulation of the ACTH-R expression in late gestation is essential for the increase in adrenal steroidogenic capacity occurring then. The data also indicate that a reduction in the ACTH-R expression blocks the ability of the peptide to stimulate early steps in the steroidogenic pathway event after maturation is complete.

Although it has been recognized for over a decade that hypothalamic-pituitary disconnection (HPD) in fetal sheep prevents the late gestation rise in plasma cortisol concentrations, the underlying mechanisms remain unclear. We hypothesized that reductions in adrenal responsiveness and ACTH receptor (ACTH-R) expression may be mediating factors. HPD or sham surgery was performed at 120 days of gestation, and catheters were placed for blood sampling. At ∼138 days of gestation, fetuses were killed, and adrenals were removed for cell culture and analyses of ACTH-R mRNA and protein. After 48 h, adrenocortical cells were stimulated with ACTH for 2 h, and the medium was collected for cortisol measurement. The same cells were incubated overnight with medium or medium containing ACTH or forskolin (FSK), followed by ACTH stimulation (as above) and cortisol and cellular ACTH-R mRNA analyses. HPD prevented the late gestation increase in plasma cortisol and bioactive ACTH and reduced adrenal ACTH-R mRNA and protein levels by over 35%. HPD cells secreted significantly less cortisol than sham cells (3.2 ± 1.2 vs. 47.3 ± 11.1 ng·ml−1·2 h−1) after the initial ACTH stimulation. Overnight incubation of HPD cells with ACTH or FSK restored cortisol responses to acute stimulation to levels seen in sham cells initially. ACTH-R mRNA levels in cells isolated from HPD fetuses were decreased by over 60%, whereas overnight incubation with ACTH or FSK increased levels by approximately twofold. Our findings indicate that the absence of the cortisol surge in HPD fetuses is a consequence, at least in part, of decreased ACTH-R expression and adrenal responsiveness.

In vitro, the growth inhibiting effect of ACTH on adrenocortical cells is well documented, even though there are reports of opposite effects under defined cell culture conditions. In vivo, activation of the ACTH receptor (ACTHR) has a trophic effect on the adrenal cortex, while the effects on proliferation are still under discussion, especially since other POMC derived peptides have been characterized. However, ACTH is thought to act as a differentiation factor with inhibiting effects on tumor growth. In undifferentiated adrenocortical carcinomas, ACTHR expression is frequently lost, which is associated with extensive tumor growth. We describe a new microsatellite marker within the intron of the ACTHR gene termed ACTHRint1. In a series of 114 patients with various adrenal and non-adrenal tumors, the rate of heterozygosity was 100 %. Only one out of 57 patients with adrenocortical adenoma showed LOH at the ACTHR locus, whereas 4 of 10 carcinomas had loss of one allele. Patients suffering from tumors with LOH showed a more aggressive disease course and had earlier recurrences with poor prognosis. These data confirm earlier findings that adrenocortical carcinomas frequently show loss of ACTHR expression, which is associated with a more aggressive tumor growth. However, whether the ACTHR is directly involved in tumor growth or acts a marker of differentiation that is lost in more advanced tumor stages is still not clear.

Familial glucocorticoid deficiency (FGD) is an autosomal recessive disorder characterized by a glucocorticoid adrenal insufficiency without mineralocorticoid deficiency. Mutations of the ACTH receptor (MC2-R) gene have been reported in some FGD cases, but only a few of them have been functionally studied. We reported clinical features and MC2-R gene analysis in three families. For each proband, an homozygous mutation was identified after amplification and sequencing of the whole intronless MC2-R gene. One mutation converted Val-142 located in the second intracellular loop to Leu. Another mutation in the sixth transmembrane domain converted Ala-233 to Pro. The last mutation converted the negatively charged Asp-103 in the first extracellular loop to an uncharged Asn. Functional studies of these mutations as well as the S120R mutation were performed after stable transfection of M3 cells and measurement of ACTH-induced cAMP production. For the S120R, V142L, and A233P mutated MC2-R, cAMP production curves were similar to that obtained with M3 parental cells, confirming that these mutations are responsible for the FGD in the affected patients. The D103N-mutated MC2-R had an impaired cAMP response to physiological doses of ACTH, but the maximal response at very high concentrations of ACTH was similar to that obtained for the wild-type MC2-R. All these results demonstrated clear relationships based on functional studies between MC2-R homozygous mutations and FGD phenotype.

We report a 30-year-old female with adrenal unresponsiveness to ACTH. Her clinical features included no adrenal crisis despite poor drug compliance, poor pubic hair development (Tanner stage 2), well-developed breasts (Tanner stage 5), and regular menstrual cycles. Endocrinological data included blood ACTH 1500 pmol/l, cortisol 18 nmol/l, dehydroepiandrosterone sulphate below 0.26 μmol/l, activated renin 0.37 pmol/l, and aldosterone 3.4 nmol/l. Direct sequencing and allele-specific amplification revealed two novel mutations in the ACTH receptor gene. One was transition from guanine to adenine at nucleotide position 1002, resulting in substitution of aspartate for asparagine at codon 103, and the other was transition from cytosine to thymine at nucleotide 1104, leading to substitution of arginine for tryptophan at codon 137. The present findings lend additional credence to the notions that adrenal androgens play an important role in female pubic hair development and that ovarian development takes place independently of adrenarche.

Several mutations in the coding exon of the ACTH receptor (MC2R) gene have been reported in cases of familial glucocorticoid deficiency or FGD. However, many patients with a similar syndrome do not present any mutation in the coding region of this gene. This is the case in 11 families we have investigated. Patients in these families present the typical clinical features of FGD, but no mutation was found in the coding exon of the ACTH receptor. To determine whether mutations on MC2R gene, but outside the coding region, may be involved in FGD in these families, we have performed a linkage analysis. Using three markers flanking MC2R gene on chromosome 18, we were able to exclude linkage in a region of 12 centimorgans around the gene. This result clearly indicates that FGD is genetically heterogeneous. Defects in gene(s) different from MC2R gene are implicated in this syndrome.

Stimulation of steroidogenesis in the adrenal cortex is a major physiological action of adrenocorticotropic hormone (ACTH). This action is presumed to be mediated by the ACTH receptor and is functionally connected with the hypothalamus-pituitary-adrenal axis. To gain information concerning the distribution of the ACTH receptor in this axis, we examined mRNA for the ACTH receptor in the adrenal gland, pituitary and hypothalamus of the mouse, by using in situ hybridization. The Y-1 mouse adrenal tumour cell line was also examined. The specific hybridization signal for ACTH receptor mRNA was uniformly distributed in the Y-1 cells, although the level of expression was low. The adrenal cortex showed a strong signal for the ACTH receptor mRNA in both the zona fasciculata and the zona glomerulosa, sites that are involved in the mediation of the action of ACTH on the synthesis and release of glucocorticoids and aldosterone. In the zona reticularis of the cortex, a small number of cells showed positive hybridization signals. Moreover, a few scattered cells were positive in the adrenal medulla. In contrast, the hybridization results for both the hypothalamus and the pituitary proved to be negative. These results indicate that the mouse ACTH receptor is a peripheral receptor that is exclusively located in the adrenal gland.

Objective Adrenocorticotropin hormone (ACTH) has been the standard treatment to infantile spasms (IS). However, the mechanism of ACTH therapy is still unclear. ACTH exerts the function via melanocortin 2 receptor (MC2R). Our previous study showed a common 4-single nucleotide polymorphism (SNP) haplotype TCCT at the MC2R promoter was strongly associated with responsiveness to ACTH therapy, where these 4 SNPs [rs1893219, rs1893220, rs2186944, and a novel SNP (T>C)] were mapped at position -853, -759, -7, and -2 bp based on the transcription start site of the MC2R gene. In this study, we further elucidated functional significances of the TCCT haplotype. Methods To evaluate whether the TCCT haplotype influences MC2R transcription levels, the luciferase reporter vector was used by a transient transfection. Expression of rat MC2R cDNA driven by the TCCT-carrying or TCCC-carrying promoter was detected by the real-time quantitative reverse transcription-PCR. These assays were performed on cell lines cultured in absence or presence of ACTH. Results In the baseline, the light intensity of the luciferase reporter assay driven by the TCCT promoter was four times higher than that by the TCCC promoter. The intensity was dramatically increased in the pGL3-TCCT after ACTH stimulation, compared to that in the pGL3-TCCC. MC2R expression assay showed a 5-fold increase in the TCCT promoter in presence of ACTH, compared with that in absence of ACTH. Conclusion The results showed that the haplotype TCCT in MC2R promoter significantly led to increased MC2R expression and strong responses to ACTH, providing evidence of the molecular mechanism of ACTH therapy in IS.

Vasoactive intestinal peptide (VIP) concentration-dependently enhanced corticosterone and cyclic-AMP release by dispersed rat inner adrenocortical cells. A VIP-receptor antagonist and the ACTH-receptor antagonist corticotropin-inhibiting peptide annulled both adrenocortical-cell responses to VIP, while the protein kinase (PKA) inhibitor H-89 blocked only corticosterone response. Collectively, these findings suggest that VIP stimulates glucocorticoid secretion of rat adrenals, through the aspecific activation of ACTH receptors coupled with the adenylate cyclase/PKA-dependent signaling pathway.

Two mutant clones (Y6 and OS3) derived from the ACTH-responsive Y1 mouse adrenocortical tumor cell line fail to respond to ACTH with increased adenylyl cyclase activity and, as a consequence, are resistant to the steroidogenic effects of the hormone. As determined from Northern blot and RNase protection assays, ACTH resistance in these mutants results from the failure to accumulate ACTH receptor transcripts. The ACTH receptor gene appears to be present in these mutants as determined by Southern blot hybridization analysis and can be activated following the growth of the mutant cells as tumors in mice, suggesting that the ACTH receptor gene is modified in a reversible manner. When mutant cells are transformed with a gene encoding the mouse beta 2-adrenergic receptor they respond to beta-adrenergic agonists with increased adenylyl cyclase activity in a manner that is indistinguishable from a similarly transformed parent Y1 cell line. These results suggest that the adenylyl cyclase system in the mutants is otherwise intact and that the failure to express ACTH receptor transcripts limits the responsiveness of these clones to the hormone.

We report a 30-year-old female with adrenal unresponsiveness to ACTH. Her clinical features included no adrenal crisis despite poor drug compliance, poor pubic hair development (Tanner stage 2), well-developed breasts (Tanner stage 5), and regular menstrual cycles. Endocrinological data included blood ACTH 1500 pmol/l, cortisol 18 nmol/l, dehydroepiandrosterone sulphate below 0.26 micromol/l, activated renin 0.37 pmol/l, and aldosterone 3.4 nmol/l. Direct sequencing and allele-specific amplification revealed two novel mutations in the ACTH receptor gene. One was transition from guanine to adenine at nucleotide position 1002, resulting in substitution of aspartate for asparagine at codon 103, and the other was transition from cytosine to thymine at nucleotide 1104, leading to substitution of arginine for tryptophan at codon 137. The present findings lend additional credence to the notions that adrenal androgens play an important role in female pubic hair development and that ovarian development takes place independently of adrenarche.

Although it is known that stresses on females damage oocytes with increased production of stress hormones, whether corticotrophin-releasing hormone (CRH) or adrenocorticotropic hormone (ACTH) harm oocytes directly are largely unknown. We demonstrated that CRH exposure during in vitro maturation impaired competence of both pig and mouse cumulus-oocyte-complexes (COCs), and it impaired competence and induced apoptosis in pig cumulus-denuded oocytes (DOs) but not in mouse DOs. CRH receptor 1 was expressed in pig DOs and in cumulus cells (CCs) of both species but not in mouse DOs. In the presence of CRH, whereas mouse CCs underwent apoptosis, pig CCs did not. While pig CCs did, mouse CCs did not express CRH-binding protein. ACTH did not affect competence of either pig or mouse COCs or DOs although they all expressed ACTH receptor. Both pig and mouse CCs expressed steroidogenic acute regulatory protein (StAR), and ACTH enhanced their progesterone production while alleviating their apoptosis. Neither pig nor mouse DOs expressed StAR, but ACTH inhibited maturation-promoting factor and decelerated meiotic progression of DOs suggesting activation of protein kinase A (PKA). In conclusion, CRH impaired pig and mouse oocyte competence by interacting with CRH receptor and inducing CCs apoptosis, respectively. ACTH activated PKA in both DOs and CCs although it showed no effect on oocyte competence.

Retinoids play a pivotal role in adrenal development and differentiation. Recent clinical trials revealed therapeutic potential of both all-trans and 9-cis retinoic acid in patients with cortisol excess due to a pituitary ACTH-secreting adenoma and indicated that retinoids might act also on the adrenal. Aim of the present study was to evaluate the effect of 9-cis retinoic acid on adrenals from patients with ACTH-dependent Cushing’s syndrome. Adrenal specimens from six patients with Cushing’s disease were incubated with 10 nM–1 µM 9-cis retinoic acid with and without 10 nM ACTH. Cortisol secretion was measured by immunoassay and expression of genes involved in steroidogenesis as well as retinoic acid action were evaluated by real-time RT-PCR. Incubation with 10–100 nM 9-cis retinoic acid increased spontaneous cortisol secretion and expression of STAR and CYP17A. On the other hand, in wells treated with ACTH, 9-cis retinoic acid markedly diminished ACTH receptor upregulation and no stimulatory effect on cortisol secretion or steroidogenic enzyme synthesis was observed. ACTH itself increased ligand-induced retinoic acid receptor expression, possibly enhancing sensitivity to retinoic acid. Our findings indicate that the effect of 9-cis retinoic acid in presence of ACTH is distinct from unchallenged wells and support the hypothesis of a direct adrenal action in patients with Cushing’s disease.

Objectives Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder characterised by isolated glucocorticoid deficiency. Melanocortin receptor 2 (MC2R) mediates the functions of adrenocorticotropic hormone (ACTH) in the adrenal cortex. MC2R accessory protein (MRAP) is a transmembrane protein involved in the trafficking of MC2R to the cell surface. Mutations in MC2R and MRAP genes cause FGD type 1 and 2. In the present case series, we evaluate the clinical characteristics and long-term follow-up of six cases with FGD due to mutations in MC2R and MRAP. Case presentation Data of six cases with FGD (five with mutations in MC2R and one with a mutation in MRAP) who were being followed at our paediatric endocrine centre was evaluated. Diagnosis of FGD was considered in case of elevated ACTH and inappropriately low cortisol level, and exclusion of other aetiologies. The main presenting complaints were hyperpigmentation and hypoglycaemic convulsion in all cases. During a follow-up period of 26–115 months, one patient with homozygous 560delT mutation in MC2R, one female with G226R mutation in MC2R and one female with IVS3ds+1delG mutation in MRAP had a neurodevelopmental delay (NDD), while the other three patients had normal neurodevelopment. Conclusions FGD patients due to MC2R and MRAP mutations with early diagnosis and compliance to the hydrocortisone therapy had normal neurodevelopment, while delay in diagnosis and poor compliance was associated with severe hypoglycaemic convulsions and subsequent complications NDD.

Objectives Primary bilateral macronodular adrenal hyperplasia is an uncommon cause of endogenous Cushing's syndrome characterized by the presence of aberrant adrenal expression of ectopic receptors that regulate steroidogenesis by mimicking the events triggered by ACTH receptor activation. Receptors of this type have been described for several hormones. The aim of the study is to detect these receptors in two patients with ACTH-independent hypercortisolism by means of the application of a screening protocol. Design and methods A protocolized study of aberrant receptors was performed including measurements of ACTH, cortisol and other steroids and hormones. Upright posture test, mixed food and administration of Gonadotropin-Releasing Hormone (GnRH) were used as stimuli. In both patients, a stimulation test with intravenous ACTH was conducted to determinate the cortical response capacity. The study was carried out in three separate days. Results The first patient, who had a hypergonadotropic hypogonadism , presented anomalous cortisol response to the GnRH stimulation, with potential medical treatment by the use of exogenous testosterone . In the second case, the patient with clinical Cushing's syndrome presented anomalous cortisol response to standing, whose potential medical treatment would be the use of beta-blockers . Conclusions This etiological variant of ACTH-independent Cushing's syndrome leads to the use of specific pharmacologic therapies in some cases as alternatives to adrenalectomy . The studied cases show the importance of having a high degree of suspicion when diagnosing less frequent types of Cushing's syndrome.

Maternal stress can impair foetal development and program sex-specific disease outcomes in offspring through the actions of maternally produced glucocorticoids, predominantly corticosterone (Cort) in rodents. We have demonstrated in mice that male but not female offspring prenatally exposed to Cort (33 µg/kg/h for 60 h beginning at E12.5) develop cardiovascular/renal dysfunction at 12 months. At 6 months of age, renal function was normal but male offspring had increased plasma aldosterone concentrations, suggesting that altered adrenal function may precede disease. This study investigated the long-term impact of prenatal exposure to Cort on adrenal growth, morphology and steroidogenic capacity as well as plasma Cort concentrations in offspring at postnatal day 30 (PN30), 6 months and 12 months of age. Prenatal Cort exposure decreased adrenal volume, particularly of the zona fasciculata, in male offspring at PN30 but increased both relative and absolute adrenal weight at 6 months of age. By 12 months of age, male Cort-exposed offspring had reduced absolute adrenal weight in association with increased adrenal plaque deposition (lipogenic pigmentation). Plasma Cort concentrations were elevated in male 6-month offspring but not at other ages. mRNA expression of Mc2r (ACTH receptor) was increased in males at PN30, and Cyp11a1 expression was decreased at 6 and 12 months of age. There were no changes in the adrenals of female Cort-exposed offspring. This study demonstrates that prenatal Cort exposure induces offspring adrenal gland dysfunction in an age- and sex-specific manner, which may contribute to long-term programmed disease in male offspring after maternal stress.

Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disease resulting from resistance to the action of adrenocorticotropic hormone (ACTH) on the adrenal cortex, which leads to isolated glucocorticoid deficiency with normal mineralocorticoid secretion. It may present in infancy or early childhood with hyperpigmentation, failure to thrive, recurrent infections, hypoglycemic attacks and convulsions that may result in coma or death. Laboratory investigations reveal low cortisol and androgen levels with high ACTH associated with normal reninaldosterone axis. The disorder may be caused by mutations in the gene of ACTH receptor (MC2R), or mutations in the newly described melanocortin− 2 receptor accessory protein (MRAP) namely, FGD type 1 and FGD type 2, respectively. Twenty five percent of FGD cases are due to the mutations of the ACTH receptor, while FGD type 2 accounts for approximately 15−20% of FGD cases. Here, we report a six−month−old male infant, who presented with recurrent hypoglycemic convulsions. Serum hormone analysis showed low cortisol and androgen levels associated with a high ACTH concentration. No mutation was found in the NR0B1 and MC2R genes excluding congenital adrenal hypoplasia and FGD type 1. We found a homozygous deletion (c. 106+1delG) in intron 3 of MRAP gene. To our knowledge, this is the first Turkish patient reported with FGD type 2 due to a known MRAP mutation. Conflict of interest:None declared.

Corticotropin releasing hormone (CRH) has been detected in the adrenal gland of many species and may be involved in regulation of glucocorticoid secretion. In cultured human fetal adrenal definitive/transitional zone cells, CRH upregulates the adrenocorticotropic hormone (ACTH) receptor and steroidogenic enzymes and is blocked by the selective CRH type 1 receptor (CRH(1)) antagonist, antalarmin. Based on these findings and evidence that antalarmin infusion into sheep suppressed prepartum increases in cortisol, we hypothesized that antalarmin would influence adrenal cortisol secretion. Antalarmin strongly attenuated ACTH and forskolin (FSK)-stimulated cortisol and cyclic adenosine monophosphate (cAMP) release from cultured ovine adrenocortical cells but did not prevent ACTH binding to cells or ACTH-induced proliferation in adult cells. Corticotropin releasing hormone was minimally effective as a secretagogue but increased the cortisol response to subsequent ACTH. These results suggest that antalarmin attenuates ACTH-induced cortisol secretion from cultured ovine adrenal cortical cells at a site distal to the ACTH receptor. Although CRH may modulate the secretory response to ACTH, it is probably not a direct cortisol secretagogue in the sheep.

Studies have indicated significant pubertal-related differences in hormonal stress reactivity. We report here that prepubertal (30 days) male rats display a more protracted stress-induced corticosterone response than adults (70 days), despite showing relatively similar levels of adrenocorticotropic hormone (ACTH). Additionally, we show that adrenal expression of the ACTH receptor, melanocortin 2 receptor ( Mc2r ), is higher in prepubertal compared to adult animals, and that expression of melanocortin receptor accessory protein ( Mrap ), a molecule that chaperones MC2R to the cell surface, is greater in prepubertal males following stress. Given that these data suggest a pubertal shift in adrenal sensitivity to ACTH, we directly tested this possibility by injecting prepubertal and adult males with 6.25 or 9.375 μg/kg of exogenous rat ACTH and measured their hormone levels 30 and 60 min post-injection. As these doses resulted in different circulating levels of ACTH at these two ages, we performed regression analyses to assess the relationship between circulating ACTH and corticosterone concentrations. We found no difference between the ages in the correlation between ACTH and corticosterone levels at the 30 min time point. However, 60 min following the ACTH injection, we found prepubertal rats had significantly higher corticosterone concentrations at lower levels of ACTH compared to adults. These data suggest that prolonged exposure to ACTH leads to greater corticosterone responsiveness prior to puberty, and indicate that changes in adrenal sensitivity to ACTH may, in part, contribute to the protracted hormonal stress response in prepubertal rats.

AimsRetinoic acid has recently yielded promising results in the treatment of Cushing's disease, i.e., excess cortisol secretion due to a pituitary corticotropin (ACTH)-secreting adenoma. In addition to its effect on the tumoral corticotrope cell, clinical results suggest an additional adrenal site of action. Aim of this study was to evaluate whether retinoic acid modulates cortisol synthesis and secretion by human adrenals in vitro.Main methodsPrimary cultures from 10 human adrenals specimens were incubated with 10nM, 100nM and 1μM retinoic acid with and without 10nM ACTH for 24h. Cortisol levels were measured by radioimmunoassay and CYP11A1, STAR and MC2R gene expression analyzed by real-time PCR.Key findingsRetinoic acid increased cortisol secretion (149.5±33.01%, 151.3±49.45% and 129.3±8.32% control secretion for 10nM, 100nM and 1μM respectively, p

ObjectiveACTH resistance syndromes are rare, autosomal, and genetically heterogeneous diseases that include familial glucocorticoid deficiency (FGD) and triple A syndrome. FGD has been shown to segregate with mutations in the gene coding for ACTH receptor (MC2R) or melanocortin 2 receptor accessory protein (MRAP), whereas mutations in the triple A syndrome (AAAS, Allgrove syndrome) gene have been found in segregation with triple A syndrome. We describe the clinical findings and molecular analysis ofMC2R,MRAP, andAAASgenes in five Brazilian patients with ACTH resistance syndrome.Design and methodsGenomic DNA from patients and their unaffected relatives was extracted from peripheral blood leucocytes and amplified by PCR, followed by automated sequencing. Functional analysis was carried out using Y6 cells expressing wild-type and mutant MC2R.ResultsAll five patients showed low cortisol and elevated plasma ACTH levels. One patient had achalasia and alacrima, besides the symptoms of adrenal insufficiency. The molecular analysis of FGD patients revealed a novel p.Gly116Val mutation in theMC2Rgene in one patient and p.Met1Ile mutation in theMRAPgene in another patient. Expression of p.Gly116Val MC2R mutant in Y6 cells revealed that this variant failed to stimulate cAMP production. The analysis of theAAASgene in the patient with triple A syndrome showed a novel g.782_783delTG deletion. The molecular analysis of DNA from other two patients showed no mutation inMC2R,MRAP, orAAASgene.ConclusionsIn conclusion, the molecular basis of ACTH resistance syndrome is heterogeneous, segregating with genes coding for proteins involved with ACTH receptor signaling/expression or adrenal gland development and other unknown genes.

Metformin is recommended as one of the first-line drugs for the treatment of type 2 diabetes and the metabolic syndrome. In addition to its insulin sensitizing effects, it has been shown to attenuate androgen excess in women with polycystic ovary syndrome (PCOS) or congenital adrenal hyperplasia (CAH), as well as to ameliorate obesity. The mechanisms of metformin action seem manifold. Preclinical studies suggest that it inhibits the cellular stress response at the level of the mitochondrial OXPHOS system and through AMPK dependent and independent mechanisms. Recent studies have shown that metformin decreases ACTH secretion from pituitary and reduces ACTH-stimulated adrenal secretion. In this study we investigated its specific effect through the melanocortin receptor 2 (MC2R) on signaling targeting adrenal steroidogenesis. To assess this effect, we used mouse adrenal OS3 cells, which do not express the MC2R. Cells were transfected with the MC2R and stimulated by ACTH. Downstream cyclic AMP production was then assessed by a co-transfected cAMP-responsive vector producing luciferase that was measured by a dual luciferase assay. The amount of luciferase produced in this assay corresponds to the amount of receptor activation with varying amount of ACTH. The effect of metformin was then tested in this system. We found a significant inhibition of ACTH induced MC2R activation and signaling with 10 mM metformin. The ACTH concentration response curve (CRC) was half-log shifted and a ∼30 % reduction in maximum receptor response (Rmax) to ACTH in presence of metformin was observed. This effect was dose dependent with an IC50 of 4.2 mM. qRT-PCR analyses showed that metformin decreased ACTH induced MC2R expression. Metformin did not affect cell viability and basal cAMP levels. We also tested the effect of metformin on homologous melanocortin receptors (MCRs). No significant effect was found on MC1R and MC4R activity. However, a log shift of EC50 of ACTH stimulation on MC3R was observed with metformin treatment. Metformin also inhibited melanocortin stimulating hormone (αMSH) induced MC3R activity. In conclusion, we show that metformin acts on MC2R and MC3R signaling directly. The role of MC2R for steroidogenesis is well established. MC3R is involved in energy balance and seems to act as a rheostat when the metabolism is challenged. Our study may explain how metformin helps in weight loss and attenuates the excess response to ACTH in androgen excess disorders such as PCOS and CAH.

We found that the tritium-labeled synthetic ACTH-like octapeptide leucocorticotropin corresponding to the 81–88 sequence of the precursor of human interleukin-1α ([3H]GK VLKKRR) is bound by the ACTH receptor of rat adrenal cortex with a high affinity and specificity (K d 2.2 ± 0.1 nM). This peptide was shown to exert no effect on the adenylate cyclase activity of the membranes of rat adrenal cortex in the concentration range from 1 to 1000 nM. Leucocorticotropin administration three times at doses of 10–20 μg/animal did not change the level of hydroxycorticosteroids (11-HOCS) in the rat adrenal glands in the absence of temperature action. At the same time, the peptide abolishes (at a dose of 20 μg/animal, three times) or significantly decreases (at a dose of 10 μg/animal, three times) the dramatic increase in the 11-HOCS content in the adrenal glands occurring in the case of cold or heat shock. Thus, leucocorticotropin normalizes the 11-HOCS level in the rat adrenal cortex during stress. The stress-protective effect of the peptide is mediated through the ACTH receptor.

Maturation of adrenocortical function is important because a prepartum increase in fetal plasma glucocorticoids is required for survival after birth. Adrenal maturation may include alterations in the regulation of adrenocorticotropic hormone (ACTH) receptor expression. Therefore, we quantitated ACTH receptor expression in the ovine adrenal cortex during development and after manipulations to better understand the regulation of the adrenal receptor in vivo. For the ontogeny study, adrenals were obtained from fetuses at different stages of development, and the cortical tissue was stored at —80C until total RNA was extracted. The ACTH binding studies were done on adrenal membranes obtained from fetuses at two different ages using I (Phe-2, Nle-4) ACTH as the ligand. Plasma ACTH was measured by two-site immunoradiometric assay, and Cortisol was measured by radioimmunoassay. ACTH receptor mRNA was quantitated by ribonuclease protection assay. The data were analyzed by analyses of variance. ACTH receptor mRNA level progressively increased in fetal life; relative changes in receptor mRNA and binding were similar (3.0-fold and 2.4-fold, respectively). Physiologic increases in fetal plasma Cortisol decreased adrenal ACTH receptor mRNA concentration, and there was a strong (r = 0.76, P

Bovine adrenal zona fasciculata cells express background K+channels ( IACchannels) whose activity is potently inhibited by ACTH. In whole cell patch clamp recordings, it was discovered that the trivalent lanthanides (Ln3+s) lanthanum and ytterbium interact with two binding sites to modulate K+flow through these channels. Despite large differences in ionic radii, these Ln3+s inhibited IACchannels half-maximally with IC50values near 50 μM. In addition, these Ln3+s blocked and reversed ACTH-mediated inhibition of IACK+channels at similar concentrations. The Ln3+s did not alter inhibition of IACby angiotensin II or cAMP. Ln3+-induced uncoupling of ACTH receptor activation from IACinhibition was prevented by raising the external Ca2+concentration from 2 to 10 mM. The divalent cation Ni2+(500 μM) also blocked ACTH-dependent inhibition of IACthrough a Ca2+-sensitive mechanism. The results are consistent with a model in which Ln3+s produce opposing actions on IACK+currents through two separate binding sites. In addition to directly inhibiting IAC, Ln3+s (and Ni2+) bind with high affinity to a Ca2+-selective site associated with the ACTH receptor. By displacing Ca2+from this site, Ln3+s prevent ACTH from binding and accelerate its dissociation. These results identify Ln3+s as a relatively potent group of noncompetitive ACTH receptor antagonists. Allosteric actions of trivalent and divalent metal cations on hormone binding, mediated through Ca2+-specific sites, may be common to a variety of peptide hormone receptors.

Adrenocorticotropin hormone (ACTH) exerts trophic effects on adrenocortical cells. We studied the phosphorylation of mitogen-activated proteins kinases (MAPKs) in human embryonic kidney cells stably expressing the ACTH receptor, MC2R, and its accessory protein MRAPβ and in primary cultures of human adrenal fasciculata cells. ACTH induced a maximal increase in p44/p42(mapk) and of p38 MAPK phosphorylation after 5min. Neither the overexpression of wild-type arrestin2, arrestin3 or their respective dominant negative forms affected p44/p42(mapk) phosphorylation. However, preincubation with the recycling inhibitors brefeldin A and monensin attenuated both cAMP accumulation and p44/p42(mapk) phosphorylation proportionally. Cyclic AMP-related PKA inhibitors (H89, KI(6-22)) and Rp-cAMPS decreased p44/p42(mapk) phosphorylation but not ACTH-mediated cAMP production. The selective Epac1/2 activator, 8-pCPT-2'-O-MecAMP, did not modify the effect of ACTH. Thus, cAMP/PKA, but not cAMP/Epac1/2 pathways, or arrestin-coupled internalization of MC2R is involved in ACTH-induced p44/p42(mapk) phosphorylation by human MC2R. Together, ACTH binding to MC2R stimulates PKA-dependent p44/p42(mapk) phosphorylation.

Familial glucocorticoid deficiency (FGD) or hereditary unresponsiveness to adrenocorticotropin (ACTH) is an autosomal recessive disorder characterized by isolated glucocorticoid deficiency associated with normal mineralocorticoid secretion. Mutations in genes encoding either ACTH receptor or melanocortin 2 receptor accessory protein are responsible for the disease in about 50% of cases, named FGD type 1 and type 2, respectively. Patients may present with hyperpigmentation, recurrent infections, failure to thrive, hypoglycemic seizures, and coma in infancy or early childhood. Here we report the case of a 17-day-old newborn diagnosed with FGD type 1 who presented with hyperbilirubinemia and hyperpigmentation, a sign which was erroneously assumed to be due to prolonged phototherapy by the referring physician. Hormone analysis showed low cortisol and high ACTH levels with normal serum electrolytes and renin-aldosterone axis. Genetic analysis revealed a novel homozygous melanocortin 2 receptor mutation p.Leu225Arg in the patient. The healthy parents were heterozygous for the mutation. Copyright (C) 2011 S. Karger AG, Basel

OBJECTIVE Familial glucocorticoid deficiency (FGD) has frequently been associated with tall stature in affected individuals. The clinical, biochemical and genetic features of five such patients were studied with the aim of clarifying the underlying mechanisms of excessive growth in these patients. PATIENTS AND METHODS Five patients with a clinical diagnosis of FGD are described in whom the disorder resulted from a variety of novel or previously described missense or nonsense mutations of the ACTH receptor (MC2-R). All patients demonstrated excessive linear growth over that predicted from parental indices and increased head circumference. RESULTS Growth hormone and IGF-I-values were normal. Growth charts suggest that the excessive growth is reduced to normal following the introduction of glucocorticoid replacement. A characteristic facial appearance including hypertelorism, marked epicanthic folds and prominent frontal bossing was noted. CONCLUSIONS These findings indicate that ACTH resistance resulting from a defective ACTH receptor may be associated with abnormalities of cartilage and/or bone growth independently of the GH–IGF-I axis, but probably dependent on ACTH actions through other melanocortin receptors.