ACTH-stimulated Corticosterone Secretion Research Articles

Discovery of CRN04894: A Novel Potent Selective MC2R Antagonist.

A novel class of nonpeptide melanocortin type 2 receptor (MC2R) antagonists was discovered through modification of known nonpeptide MC4R ligands. Structure-activity relationship (SAR) studies led to the discovery of 17h (CRN04894), a highly potent and subtype-selective first-in-class MC2R antagonist, which demonstrated remarkable efficacy in a rat model of adrenocorticotrophic hormone (ACTH)-stimulated corticosterone secretion. Oral administration of 17h suppressed ACTH-stimulated corticosterone secretion in a dose-dependent manner at doses ≥3 mg/kg. With its satisfactory pharmaceutical properties, 17h was advanced to Phase 1 human clinical trials in healthy volunteers with the goal of moving into patient trials to evaluate CRN04894 for the treatment of ACTH-dependent diseases, including congenital adrenal hyperplasia (CAH) and Cushing's disease (CD).

Inadequate corticosterone levels relative to arthritic inflammation are accompanied by altered mitochondria/cholesterol breakdown in adrenal cortex: a steroid-inhibiting role of IL-1β in rats

ObjectivesIn rheumatoid arthritis, inadequate cortisol secretion was observed relative to inflammation, but reasons are unknown. Human adrenal glands cannot be investigated due to ethical reasons. Thus, a model of arthritis...

Expression and homologous regulation of GH secretagogue receptor mRNA in rat adrenal gland.

GH secretagogues (GHSs) elicit a variety of biological effects in several endocrine and non-endocrine target tIssues, including activation of the hypothalamic-pituitary-adrenal axis. The latter is mainly carried out through a central hypothalamic action; yet the possibility of additional effects directly at the adrenal level cannot be ruled out. The aims of this study were to evaluate the expression and homologous regulation of the GHS-receptor (GHS-R) gene in rat adrenal and to assess the effects of synthetic (GH releasing peptide-6 - GHRP-6) and natural (ghrelin) ligands of GHS-R upon basal and ACTH-stimulated corticosterone secretion in vitro. Analysis of adrenal expression of target mRNAs (GHS-R, GHS-R1a, ghrelin, and several steroidogenic factors) was conducted by means of primer-specific, semi-quantitative RT-PCR. Evaluation of corticosterone secretion by incubated adrenal tIssue was carried out by specific RIA. RT-PCR analysis demonstrated expression of the GHS-R gene, but not of the gene encoding the cognate ligand ghrelin, in rat adrenal. Moreover, expression of the mRNA coding for the type 1a GHS-R (GHS-R1a), i.e. the biologically active receptor form, was demonstrated. The adrenal expression of the GHS-R message appeared under the regulation of homologous signals in vitro, as short-term incubation of adrenal samples in serum-free medium induced a significant increase in GHS-R mRNA levels that was inhibited by exposure to different doses of GHRP-6 (10(-9)-10(-5) mol/l) or ghrelin (10(-7) mol/l). Notably, an opposite pattern of homologous regulation of GHS-R gene expression was observed at the pituitary. Finally, short-term stimulation with increasing concentrations of GHRP-6 (10(-9)-10(-5) mol/l) or ghrelin (10(-7) mol/l) failed to alter basal and ACTH-stimulated corticosterone secretion in vitro, neither did it modify ACTH-stimulated mRNA expression levels of several upstream elements in the steroidogenic route: the steroidogenic acute regulatory (StAR) protein, and the enzymes P450 cholesterol side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Our study provides novel evidence for the expression and homologous regulation of the GHS-R gene in rat adrenal. However, our results cast doubts on the possibility of direct adrenal actions of ligands of the GHS-R in the regulation of corticosterone secretion in the rat.

Homologous and heterologous down-regulation of leptin receptor messenger ribonucleic acid in rat adrenal gland.

Leptin, the adipocyte-produced hormone that plays a key role in body weight homeostasis, has recently been found to be involved in the regulation of the hypothalamic-pituitary-adrenal axis. Moreover, reciprocal interactions between leptin and glucocorticoids have been described. In the present communication, two different strategies were undertaken to explore the mode of action of leptin in the direct control of rat adrenal function. First, a synthetic peptide approach demonstrated that the inhibitory effect of leptin on basal and ACTH-stimulated corticosterone secretion in vitro is, at least partially, mapped to a domain of the native protein between amino acids 116 and 130, i.e. an area of the molecule also relevant in terms of regulation of food intake and endocrine control. Secondly, semi-quantitative RT-PCR analysis indicated a complex pattern of adrenal leptin receptor (Ob-R) mRNA expression, with predominant expression of the Ob-Ra and Ob-Rb isoforms, as well as moderate levels of the Ob-Rc and Ob-Rf variants, whereas negligible signals for the Ob-Re isoform were detected. Interestingly, such an expression pattern appeared hormonally regulated as exposure to human recombinant leptin (10(-7 )M) or ACTH (10(-7 )M) significantly decreased Ob-R isoform mRNA expression. Indeed, dose-dependent ligand-induced Ob-Ra and Ob-Rb mRNA down-regulation was further confirmed by adrenal stimulation with increasing concentrations (10(-9)-10(-5 )M) of the active leptin fragment, leptin 116-130 amide. Overall, our results provide evidence for a novel regulatory step at the level of Ob-R mRNA expression in the interplay between ACTH and leptin for the tuning of rat adrenal corticosterone secretion. Furthermore, our data showing down-regulation of Ob-R mRNA expression by its cognate ligand may well be relevant to leptin physiology and its alteration in various disease states.

Adrenocorticotropin (ACTH) and corticosterone secretion by perifused pituitary and adrenal glands from rodents exposed to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)

Although in utero maternal stress has been shown to have lasting effects on rodent offspring, fetal effects of chemically-induced alterations of the maternal hypothalamic-pituitary-adrenal axis (HPA) have not been well studied. This study examined the effects of in vivo 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) exposure on pituitary-adrenal function in the male rat, pregnant female rat and pregnant female mouse. The secretion of adrenocorticotropin (ACTH) and corticosterone (CORT) in pituitary and adrenal glands, respectively, was assessed in ex vivo perifusion cultures. Male and pregnant female (gestation day 8) Sprague–Dawley rats were gavaged once with 10 μg/kg TCDD, pregnant female mice once with 24 μg/kg TCDD, and euthanized 10 days later. Hemi-pituitary (rat) or whole anterior pituitaries (mice) and right adrenal glands from the same animal were quartered, perifused under baseline and stimulated conditions. In both males and pregnant females, TCDD did not affect corticotropin releasing hormone (CRH)-stimulated ACTH secretion. Neither total pituitary ACTH nor plasma ACTH was altered in either sex or species by TCDD treatment. ACTH-stimulated CORT secretion was not affected by TCDD in either sex or species, and adrenal tissue and plasma CORT levels were unchanged in males and pregnant females by TCDD. However, the plasma ACTH:CORT ratio was decreased about 46% in male rats treated with TCDD. Plasma CORT levels were 23-fold higher and plasma ACTH levels were 1.5-fold higher in pregnant females than in male rats. In male versus female rats, adrenal CORT and anterior pituitary ACTH tissue levels were about 7.5- and 1.75-fold higher and ACTH, respectively. Female mouse adrenal tissue CORT was about 4-fold greater than female rat. The reduced plasma ACTH:CORT ratio in the male rat suggests that TCDD disturbs HPA function. Exposure of male rat to a 5-fold higher dose in earlier studies clearly demonstrated effects of TCDD on male rat HPA. The present study identified substantial HPA performance differences between male and pregnant female rats. The failure to detect a response to TCDD in pregnant female rat and mouse could be a function of both TCDD dose and the high level of secretion of both ACTH and CORT in pregnant animals. For the rat or mouse, a single exposure to TCDD during pregnancy does not appear sufficient to induce maternally-mediated developmental, reproductive and behavioral toxicity via the HPA axis.

Acute effects of thyroid hormones on the production of adrenal cAMP and corticosterone in male rats.

The acute effects of thyroid hormones on glucocorticoid secretion were studied. Venous blood samples were collected from male rats after they received intravenous 3,5,3'-triiodothyronine (T3) or thyroxine (T4). Zona fasciculata-reticularis (ZFR) cells were treated with adrenocorticotropic hormone (ACTH), T3, T4, ACTH plus T3, or ACTH plus T4 at 37 degrees C for 2 h. Corticosterone concentrations in plasma and cell media, and also adenosine 3',5'-cyclic monophosphate (cAMP) production in ZFR cells in the presence of 3-isobutyl-1-methylxanthine, were determined. The effects of thyroid hormones on the activities of steroidogenic enzymes of ZFR cells were measured by the amounts of intermediate steroidal products separated by thin-layer chromatography. Administration of T3 and T4 suppressed the basal and the ACTH-stimulated levels of plasma corticosterone. In ZFR cells, both thyroid hormones inhibited ACTH-stimulated corticosterone secretion, but the basal corticosterone was inhibited only with T3 > 10(-10) M or T4 > 10(-8) M. Likewise, T3 or T4 at 10(-7) M inhibited the basal- and ACTH-stimulated levels of intracellular cAMP. Physiological doses of T3 and T4 decreased the activities of 3 beta-hydroxysteroid dehydrogenase, 21-hydroxylase, and 11 beta-hydroxylase. These results suggest that thyroid hormones counteract ACTH in adrenal steroidogenesis through their inhibition of cAMP production in ZFR cells.

Neuropeptide Y inhibits corticosterone secretion by isolated rat adrenocortical cells

Studies with isolated rat adrenocortical cells have shown that neuropeptide Y (NPY) inhibits both basal and ACTH-stimulated corticosterone secretion. These results suggest the regulatory role of NPY in corticosterone secretion from the adrenal gland, especially during stress.

Evidence that long-term administration of a methionine-enkephalin analogue stimulates the growth and steroidogenic capacity of rat inner adrenocortical cells

A prolonged infusion with D-ala 2-met-enkephalinamide (DALA) caused a significant increase in both basal and ACTH-stimulated corticosterone secretion by dispersed inner adrenocortical cells of rats whose hypothalamo-hypophyseal axis was pharmacologically interrupted. This effect of DALA was associated with a notable hypertrophy of isolated cells. These findings suggest that enkephalins are involved in the stimulation of the growth and steroidogenic capacity of the cells of the inner layers of rat adrenal cortex.

Etomidate suppresses adrenocortical function by inhibition of 11 beta-hydroxylation.

We studied the effect of short term infusion of the imidazole-derived anesthetic agent etomidate on plasma concentrations of ACTH, cortisol, and the cortisol-precursors 11-desoxycortisol and 17-hydroxyprogesterone. During the infusion of etomidate, a significant increase in the peripheral concentration of ACTH occurred, while plasma cortisol concentrations decreased. After the end of the infusion, cortisol concentrations further decreased, while the concentrations of desoxycortisol and 17-hydroxyprogesterone increased. Furthermore, in in vitro experiments with isolated rat pituitary and adrenal cells, etomidate did not affect corticotropin-releasing hormone-induced ACTH secretion from pituitary cells, whereas ACTH-stimulated corticosterone secretion from adrenal cells was inhibited by addition of etomidate in concentrations which occur in plasma during and after infusion of the drug. These results lead to the conclusion that etomidate inhibits adrenal 11 beta-hydroxylation.

The Met-enkephalin analog D-Ala 2-Met-enkephalinamide decreases the adrenocortical response to ACTH in dispersed rat adrenal cells

The effects of the Met-enkephalin analog D-Ala 2-Met-enkephalinamide (DALA) on basal and ACTH-stimulated corticosterone secretion from dispersed adrenal cells were investigated. Low doses (10 −10 and 10 −12 M) of DALA resulted in no apparent alteration in the response to ACTH (8×10 −9, 3.2×10 −8 or 1.6×10 −7 M). High doses of DALA (10 −8 and 10 −6 M) produced a decline in the steroidogenic response to ACTH. The opiate receptor antagonist naloxone (10 −4–10 −10 M) did not influence the basal production of corticosterone or the stimulating action exerted by ACTH. However, the presence of naloxone reversed the blocking action on corticosterone production that was exerted by DALA. These findings indicate that enkephalins may decrease adrenocortical responsiveness to ACTH.

Naloxone has a direct effect on the adrenal cortex.

The effects of naloxone on basal and ACTHstimulated corticosterone secretion from superfused rat adrenocortical tissue were investigated. High doses (10-3 and 10-4M) of naloxone produced a decline in basal steroidogenesis, whereas lower doses (10-5-10-7 M) of naloxone resulted in elevations of corticosterone secretion. The simultaneous addition of an aliquot of ACTH and varying amounts of naloxone resulted in a dose-dependent alteration in the steroidgenic response by the tissue to ACTH. With higher doses (10-3 and 10-4 M) of naloxone, there were little or no significant alterations in the response to an initial dose of ACTH; however, the steroidogenic responses to ACTH administered 2 and 4 h after the single aliquot of naloxone were significantly potentiated. The lower dose of naloxone (10-610-10 M) resulted only in the potentiation of the initial ACTH response. The simultaneous administration of ACTH and 10-12 M naloxone resulted in no apparent alteration in the response to ACTH at any period in the studies. Methionine enkephalin (1.7 × 10-9 to 1.7 × 10-7 M) significantly potentiated ACTH-stimulated steroid secretion 2 h after the enkephalin was given. Naloxone also had direct effects on the cell membrane potential (MP) of superfused adrenocortical tissue. The addition of 10-5-10-10 M naloxone resulted in a rapid depolarization of the MP. The initial depolarizations lasted for approximately 15–25 min. Action potential-like spikes were often characteristic of the MP changes. These findings clearly demonstrate a direct effect of both opioid agonist and antagonist on adrenal steroid secretion. The most prominent effect was a potentiation of the steroidogenic action of ACTH. We suggest that naloxone and possibly other opioids exert their effects on the adrenal cortex by interacting with one or more relatively nonspecific membrane receptors which are distinct from the ACTH receptor. The data also suggest that one mechanism by which the opioids might exert their potentiating effect is via an alteration in membrane permeability to one or more ions, resulting in more optimal intracellular ion concentration.

Effect of beta-endorphin on the steroid production of isolated zona glomerulosa and zona fasciculata cells

Small doses of β-endorphin (10 −11−10 −5M) decrease corticosterone production of zona fasciculata cells but fail to influence steroid production of zona glomerulosa cells. 10 −4M β-endorphin increases corticosterone production of both zones. The stimulating effect of ACTH on zona fasciculata corticosterone- and zona glomerulosa aldosterone production was decreased by β-endorphin (10 −9−10 −7M). Conclusion: β-endorphin might modulate both basal and ACTH stimulated corticosterone secretion.

Noise stress and in vitro adrenocortical responsiveness to ACTH in wild cotton rats, Sigmodon hispidus

Wild cotton rats, Sigmodon hispidus, were utilized in an attempt to clarify further the interaction of intermittent high-intensity noise with the hypothalamic-pituitary-adrenal axis. Animals were collected during 3-week periods in the spring and autumn of the year. Within seasonal groups paired adrenals from control animals and from animals exposed to recorded jet aircraft noise were either (a) incubated in the presence or absence of ACTH and analyzed for cAMP content as well as corticosterone secreted or (b) subjected to an initial incubation in the presence or absence of ACTH followed by a second incubation in the presence of ACTH after which corticosterone content of the incubation media was analyzed. During initial incubations glands from noise-exposed animals within both seasonal groups exhibited slight but insignificant elevations in basal corticosterone secretion rates as compared to their control counterparts. However, ACTH elicited a significantly smaller response in terms of corticosterone secreted in (a) the noise-exposed as compared to control groups within either season and (b) the autumn as compared to the spring control groups. Additionally ACTH induced significantly smaller cAMP accumulations in noise-exposed as compared to control animals. Analysis of ACTH-stimulated corticosterone secretion after preincubation in the presence or absence of ACTH indicated either similar significant increases in both the control and noise-exposed groups (preincubation without ACTH) or significantly greater responsiveness in the noise-exposed as compared to the control group (preincubation with ACTH). The data suggest the increase of a noise-related factor of unknown origin which has the ability to diminish the expression of ACTH upon the adrenal cortex.