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Neuromedin U (NmU) is a neuropeptide with anorexigenic activity. Two receptor subtypes (NmUR1 and NmUR2) confer the effects of NmU on target cells. We have recently demonstrated that NmU reduces insulin secretion from isolated pancreatic islets. Aim of our current study is to investigate the role of somatostatin at mediating the effects of NmU on insulin secretion.

CITED2gene deletion in mice leads to adrenal agenesis. Therefore, we analyzed CITED2, a CBP/p300 interacting transactivator with transforming activity, in the human adrenal gland. In this study, we examined CITED2 expression in human embryonic and adult adrenal glands as well as adrenocortical carcinomas. As ACTH and basic fibroblast growth factor (bFGF) are connected to the physiology and growth of adrenocortical cells we studied the regulation of CITED2 by these factors in the NCI-H295R adrenocortical carcinoma cell line. We found CITED2 expression in the adult adrenal cortex as well in adrenocortical carcinomas. At an early stage of human adrenal organogenesis CITED2 could be located to the definitive zone of the developing adrenal gland using immunohistochemistry. In NCI-H295R cells, stimulation by bFGF led to a dose-dependent increase in CITED2 promotor activity, mRNA and protein expression while ACTH had no significant effect. The stimulatory effect of bFGF could be reduced by blocking mitogen-activated protein kinase activity using the MAPkinase kinase (MEK1)-inhibitor PD98059. CITED2 is expressed in embryonic and adult human adrenal glands as well as in adrenocortical cancer. It is connected to the signaling cascades of bFGF and its expression is modulated by mitogen-activated protein kinases. This suggests a novel role for CITED2 in human adrenal growth and possibly in adrenal tumorigenesis.

Beacon gene expression is elevated in the hypothalamus of the Israeli sand rat (Psammomys obesus), an animal that is used as a polygenic animal model of obesity and NIDDM. We performed studies aimed at investigating the expression of beacon mRNA and protein in pancreatic islets of the rat and the possible beacon protein effects on insulin secretion.

Adrenal zona glomerulosa cells are provided with adrenomedullin receptors. Adrenomedullin has recently been found to enhance proliferation of cultured rat vascular smooth muscle cells and zona glomerulosa cells.

Endothelin‐1 (ET‐1)[1–31] is a novel hypertensive peptide that mimics many of the vascular effects of the classic 21 amino acid peptide ET‐1[1–21]. However, at variance with ET‐1[1–21] that enhances aldosterone secretion from cultured rat zona glomerulosa (ZG) cells by acting via ETB receptors, ET‐1[1–31] did not elicit such effect. Both ET‐1[1–21] and ET‐1[1–31] raised the proliferation rate of cultured ZG cells, the maximal effective concentration being 10−8M. This effect was blocked by the ETA‐receptor antagonist BQ‐123 and unaffected by the ETB‐receptor antagonist BQ‐788. Quantitative autoradiography showed that ET‐1[1–21] displaced both [125I]PD‐151242 binding to ETA receptors and [125I]BQ‐3020 binding to ETB receptors in both rat ZG and adrenal medulla, while ET‐1[1–31] displaced only [125I]BQ‐3020 binding. The tyrosine kinase (TK) inhibitor tyrphostin‐23 and the p42/p44 mitogen‐activated protein kinase (MAPK) inhibitor PD‐98059 abolished the proliferogenic effect of ET‐1[1–31], while the protein kinase‐C (PKC) inhibitor calphostin‐C significantly reduced it. ET‐1[1–31] (10−8M) stimulated TK and MAPK activity of dispersed ZG cells, an effect that was blocked by BQ‐123. The stimulatory action of ET‐1[1–31] on TK activity was annulled by tyrphostin‐23, while that on MAPK activity was reduced by calphostin‐C and abolished by either tyrphostin‐23 and PD‐98059. These data suggest that ET‐1[1–31] is a selective agonist of the ETA‐receptor subtype, and enhances proliferation of cultured rat ZG cells through the PKC‐ and TK‐dependent activation of p42/p44 MAPK cascade.

Endothelin-1 (ET-1)[1–31] is a novel hypertensive peptide that mimics many of the vascular effects of the classic 21 amino acid peptide ET-1[1–21]. However, at variance with ET-1[1–21] that enhances aldosterone secretion from cultured rat zona glomerulosa (ZG) cells by acting via ETB receptors, ET-1[1–31] did not elicit such effect. Both ET-1[1–21] and ET-1[1–31] raised the proliferation rate of cultured ZG cells, the maximal effective concentration being 10 −8M. This effect was blocked by the ETA-receptor antagonist BQ-123 and unaffected by the ETB-receptor antagonist BQ-788. Quantitative autoradiography showed that ET-1[1–21] displaced both [ 125I]PD-151242 binding to ETA receptors and [ 125I]BQ-3020 binding to ETB receptors in both rat ZG and adrenal medulla, while ET-1[1–31] displaced only [ 125I]BQ-3020 binding. The tyrosine kinase (TK) inhibitor tyrphostin-23 and the p42/p44 mitogen-activated protein kinase (MAPK) inhibitor PD-98059 abolished the proliferogenic effect of ET-1[1–31], while the protein kinase-C (PKC) inhibitor calphostin-C significantly reduced it. ET-1[1–31] (10 −8M) stimulated TK and MAPK activity of dispersed ZG cells, an effect that was blocked by BQ-123. The stimulatory action of ET-1[1–31] on TK activity was annulled by tyrphostin-23, while that on MAPK activity was reduced by calphostin-C and abolished by either tyrphostin-23 and PD-98059. These data suggest that ET-1[1–31] is a selective agonist of the ETA-receptor subtype, and enhances proliferation of cultured rat ZG cells through the PKC- and TK-dependent activation of p42/p44 MAPK cascade.

Although oxidation of Cortisol or corticosterone by 11β‐hydroxysteroid dehydrogenase (11β‐HSD) represents the physiological mechanism conferring specificity for aldosterone on the mineralocorticoid receptor in mineralocorticoid target tissues, little attention has been paid until now to the expression and activity of this enzyme in human adrenals. We have shown that human adrenal cortex expresses 11β‐HSD type 2 (11β‐HSD2) gene, and found a marked 11β‐HSD2 activity in microsomal preparations obtained from slices of decapsulated normal human adrenal cortices. Under basal conditions, adrenal slices secreted, in addition to cortisol and corticosterone (B), sizeable amounts of cortisone and 11‐dehydrocorticosterone (DH‐B), the inactive forms to which the former glucocorticoids are converted by 11β‐HSD. Addition of the 11β‐HSD inhibitor glycyrrhetinic acid elicited a moderate rise in the production of cortisol and B and suppressed that of cortisone and DH‐B. ACTH and angiotensin II evoked a marked rise in the secretion of cortisol and B, but unexpectedly depressed the release of cortisone and DH‐B. ACTH also lowered the capacity of adrenal slices to convert [3H]cortisol to [3H]cortisone. This last effect of ACTH was concentration‐dependently abolished by both aminoglutethimide and cyanoketone, which blocks early steps of steroid synthesis, but not by metyrapone, an inhibitor of 11β‐hydroxylase. Collectively, these findings indicate that the human adrenal cortex possesses an active 11β‐HSD2 engaged in the inactivation of newly formed glucocorticoids. The activity of this enzyme is negatively modulated by the main agonists of glucocorticoid secretion through an indirect mechanism, probably involving the rise in the intra‐adrenal concentration of non‐11β‐hydroxylated steroid hormones.—Mazzocchi, G., Rossi, G. P., Neri, G., Malendowicz, L. K., Albertin, G., Nussdorfer, G. G. 11β‐Hydroxysteroid dehydrogenase expression and activity in the human adrenal cortex. FASEB J.12, 1533–1539 (1998)

Mazzocchi, G., L. K. Malendowicz, P. Rebuffat, C. Tortorella and G. G. Nussdorfer. Arginine-vasopressin stimulates Crh and Acth release by rat adrenal medulla, acting via the V1receptor subtype and a protein kinase C-dependent pathway. Peptides 18(2) 191–195, 1997.—Arginine-vasopressin (AVP) is a hypothalamic hormone that, like CRH, stimulates the pituitary release of ACTH, thereby activating adrenal glucocorticoid secretion. Evidence indicates that rat adrenal medulla contains a CRH-ACTH system duplicating that existing at the hypothalamo-pituitary level and involved in the paracrine stimulation of the cortex secretion. Therefore, we investigated by RIA the effect of AVP on the release of CRH and ACTH immunoreactivities (IR) by rat adrenal medulla in vitro. AVP concentration-dependently enhanced the release of both CRH-IR and ACTH-IR, and the effect was blocked by a selective antagonist of the V1subtype of AVP receptors. The CRH receptor antagonist α-helical-CRH partially reversed AVP-evoked rise in ACTH-IR release, without altering either CRH response or basal secretions of CRH and ACTH. The specific inhibitors of protein kinase C Ro31-8220 and calphostin C abolished both CRH and ACTH responses to AVP. In conclusion, our present findings suggest that AVP stimulates intramedullary the CRH-ACTH system, acting via V1receptors and activating protein kinase C.

Leptin, the product of the obgene, is an adipose-tissue secreted hormone, which regulates satiety, metabolic rate and thermogenesis. Many lines of evidence suggest the existence of mutual relationships between leptin and adrenal-cortex secretion, but in vivostudies gave rather conflicting results. Therefore, we have investigated the acute effect of the systemic bolusadministration of leptin (5 or 10 nmol/kg) on the function of rat pituitary-adrenocortical function. The blood concentrations of ACTH, aldosterone and corticosterone were measured by specific RIA 60 or 120 min after the leptin injection Leptin did not affect the blood concentrations of ACTH and aldosterone at 60 min, but at 120 min the lower dose of the peptide increased them. In contrast, the blood level of corticosterone was markedly enhanced by both doses of leptin at 60 and 120 min. Collectively, these findings indicate that leptin exerts a moderate acute activation of the pituitary-adrenocortical function in rats. They also suggest that the adrenocortical secretagogue action of leptin is not only mediated by the enhanced pituitary ACTH release, but is also consequent to a direct stimulatory effect of the peptide on adrenocortical cells.

The zonation and cellular composition of the adrenal cortex of intact mature male and female rats of different strains (Chbb Thomm, CFY) and three strains of Wistar rats (W1, W2 and W3) at the age of 84–90 days were compared using morphometry.

In the hamster, the weight of the adrenal glands increases during the course of gestation, with the highest value at day 5. In comparison to non-pregnant control animals, there were no changes in the volume of the zona glomerulosa (ZG) and zona fasciculata (ZF), while the volume of the zona reticularis (ZR) increased notably. The average volume of ZG-cells rose at day 5 of pregnancy and thereafter gradually decreased to that of control hamsters. A marked drop in the volume of ZF-cells was seen at days 5 and 10 of pregnancy, whereas at day 15 the cells were larger than in controls. At day 5 of pregnancy, a conspicuous increase in the cell volume was found in ZR, followed by lower values at day 10 and again higher than in control hamsters at day 15. The total number of parenchymal cells in hamster adrenal cortex increased at day 10 of gestation, then underwent a marked decrease, reaching the control value at the final day of pregnancy; this drop was mainly due to a reduction in the number of ZF-cells. The changes in the cell volume were paralleled by rather proportional changes in the volume of the mitochondrial compartment and in the quantity of smooth endoplasmic reticulum. The volume of the lipid-droplet compartment significantly rose in the course of gestation in both ZF- and ZR-cells. The cortisol output by adrenal homogenates gradually decreased during pregnancy.

Endothelins (ET) are a family of vasoactive peptides that act via two subtypes of receptors, named ETA and ETB. ET-1 binds to both ETA and ETB, whereas the isopeptide ET-3 preferentially binds to ETB. The localization of ETA and ETB receptors in the rat adrenal gland and their involvement in the adrenal secretagogue effect of ETs has been studied in vitro. Autoradiographic assessment of the selective displacement of [¹²⁵I]ET-1, [¹²⁵I]ET-3 and [¹²⁵I]BQ-3020 (an ETB agonist) by BQ-123 or BQ-788 (specific antagonists of ETA and ETB, respectively) indicates that the zona glomerulosa and adrenal medulla possess both ETA and ETB, whereas the zona fasciculata/reticularis is exclusively provided with ETB. ET-1, ET-3 and BQ-3020 enhance aldosterone and corticosterone secretion by dispersed cells of the zona glomerulosa and zona fasciculata/reticularis, respectively. BQ-123 does not affect the secretagogue action of these three agonists, whereas BQ-788 completely annuls it. ET-1 induces a marked rise in catecholamine release by fragments of the adrenal medulla, and both BQ-123 and BQ-788 partially reverse this effect. ET-3 and BQ-3020 elicit a catecholamine release that is less intense than that produced by ET-1; this response is unaffected by BQ-123 and abolished by BQ-788. Thus, in the rat, the corticosteroid secretagogue effect of ETs seems to be exclusively mediated by the ETB receptor subtype, and the catecholamine secretagogue action by both ETA and ETB. The functional relevance of ETA receptors present in the zona glomerulosa remains to be investigated.

Adult female rats have heavier adrenals than males. Orchiectomy increases the adrenal weight, an effect reversed by testosterone. Neither ovariectomy nor estradiol replacement has an effect on absolute adrenal weight.

Adrenal glands of adult female rats are heavier than the glands of corresponding male rats. Postpubertal orchiectomy increases the adrenal weight, an effect restored by testosterone replacement. Under the same conditions ovariectomy of 8 weeks duration does not change the adrenal weight while estradiol replacement enhances the relative adrenal weight.

Adult male rats were treated daily for 18 days with endoxan in doses of 4 mg/kg of body weight or testosterone in doses 2.5 mg per rat. For the last 5 days of the experiment some of the rats received gonadotrophin injections in doses of 50 I.U. Karyometric and stereologic studies were undertaken on paraffin sections of the testes stained with haematoxylin and eosin.

The effects of a prolonged (30-day) treatment with daily therapeutical doses of cyclosporin A (CAS) (20 mg/kg) on testicular Leydig cells were studied in adult rats. CSA administration provoked a significant decrease in both basal and human chorionic gonadotropin (hCG)-stimulated testosterone concentration in the peripheral blood without affecting the volume of the testes or the interstitial space. However, there was conspicuous atrophy of the Leydig cells, due mainly to a decrease in mitochondria and smooth endoplasmic reticulum, the organelles containing the enzymes of testosterone synthesis. Lipid droplets, in which cholesterol is stored, were notably increased. The nuclear volume and the surface area per cell of rough endoplasmic reticulum fell significantly in Leydig cells of CAS-treated animals. In light of these findings, it is concluded that CSA inhibits the growth and steroidogenic capacity of rat Leydig cells, probably by depressing their protein synthesis. Whether the mechanism underlying the action of CSA on Leydig cells is only indirect, by blockade of hypophyseal gonadotropin release, or also direct is unsettled and requires further investigation.

CRH and ACTH immunoreactivities (ir) were present in rat adrenal glands but not in adrenocortical autotransplants lacking chromaffin cells. Interleukin-1β (IL-1β) dose-dependently elicited CRH-ir and ACTH-ir release by decorticated adrenal fragments mainly composed of zona-medullaris tissue; the minimum effective concentration was 10^-10/10^-8 M, and the maximal one was 10^-6 M. The IL-1β (10^-6 M)-induced ACTH release by our preparations was completely blocked by α-helical-CRH (10^-6 M), a competitive inhibitor of CRH. These findings suggest that chromaffin medullary cells of the rat adrenals contain a CRH/ACTH system, duplicating that operating at the hypothalamohypophyseal level, which is stimulated by IL-1β. Thus, the mechanism underlying the well-known glucocorticoid secretagogue effect of interleukins may involve the activation of both the central and the peripheral branch of the hypothalamohypophyseal-adrenal axis. Copyright 1993, 1999 Academic Press

The effect of an 1p. bolus injection of 200 μg kg−1bacterial lipopolysaccharide (LPS) on the plasma concentrations of ACTH and corticosterone (B) were studied in intact and hypophysectomized/ACTH replaced (Hx) rats. Hormonal blood levels were measured by RIA, 30, 60, 120, 180 and 240 min after the injection. The stress evoked by the vehicle i.p injection provoked significant rises in ACTH and B blood levels at 30 and 60 min in intact rats, but not in Hx animals. In intact rats, LPS enhanced (over the respective control value) ACTH plasma level at 60, 120 and 180 min, and B plasma concentration at 120, 180 and 240 min. In Hx rats, LPS did not affect ACTH blood level, but raised B plasma concentration at 60, 120 and 180 min. B response to LPS at 120 min was completely annulled, in both intact and Hx rats, by the simultaneous administration of 25 nmol · kg−1α-helical-CRH and corticotropin-inhibiting peptide that are competitive inhibitors of CRH and ACTH, respectively. The hypothesis is advanced that LPS may activate hypothalamo-pituitary adrenal axis in rats, by stimulating not only the central (hypothalamo-pituitary), but also the peripheral (intra-adrenal) branch of the CRH/ACTH system

The cytological aspects of sexual dimorphism in the rat adrenal cortex and its relationship to the gonads have been investigated. The adrenal glands of mature female rats were heavier than those of males, and morphometry showed that this was almost exclusively due to conspicuous differences in the volume of cells of the zona fasciculata (ZF) and zona reticularis (ZR). Stereology demonstrated that the volume of the mitochondrial and lipid droplet compartments, as well as the surface area per cell of mitochondrial cristae and smooth endoplasmic reticulum tubules, were markedly higher in the ZF and ZR cells of female animals. Orchiectomy increased and ovariectomy decreased the adrenal weight, by eliciting hypertrophy and atrophy, respectively, of ZF and ZR cells; these effects of gonadectomy were reversed by the appropriate gonadal hormone replacement. It is suggested that the sexual dimorphism of the rat adrenal cortex may depend upon the inhibitory action of testosterone and the stimulatory effect of estradiol on the hypothalamo-hypophyseal-adrenal axis.

The morphology and function of isolated inner (zona fasciculata/reticularis) adrenocortical cells of rats pretreated with ACTH for 3, 6, 9 or 12 days were investigated. ACTH treatment induced a notable time-dependent enhancement in the steroidogenic capacity (corticosterone production) and growth of inner cells. The volumes of cells, mitochondrial compartment, membrane space [the cellular space occupied by smooth endoplasmic reticulum (SER) membranes] and lipid-droplet compartment, as well as the surface area of mitochondrial cristae and SER tubules, were increased in relation to the duration of ACTH pretreatment, and showed a highly significant positive linear correlation with both basal and stimulated corticosterone production. The acute exposure of isolated cells to ACTH provoked a striking lipid-droplet depletion, the extent of which was linearly and positively correlated with stimulated corticosterone secretion. The hypertrophy of the mitochondrial compartment and SER are interpreted as the morphological counterpart of the enhanced steroidogenic capacity of inner adrenocortical cells, inasmuch as the enzymes of steroid synthesis are located in these two organelles, and it is well known that chronic ACTH exposure stimulates the de novo synthesis of many of them in vivo. The rise in the number of lipid droplets, in which cholesterol is stored, is interpreted as being due to the fact that, under chronic ACTH treatment, the processes leading to cholesterol accumulation in adrenocortical cells (exogenous uptake and endogenous synthesis) exceed those of its utilization in basal steroid secretion. Cholesterol accumulated in lipid droplets as a reserve material may be rapidly utilized after acute ACTH exposure to meet the needs of the enhanced steroidogenic capacity of adrenocortical cells.

The morphological counterpart of the well-known age-dependent marked impairment of glucocorticoid secretion of rat adrenals was investigated by use of morphometric techniques. For this purpose 4-, 8-, 16- and 24-month-old rats were studied. Despite the notable lowering of both basal and ACTH-stimulated production of corticosterone by collagenase-dispersed inner adrenocortical cells, ACTH and corticosterone plasma concentrations displayed significant increases with ageing. Zona fasciculata (ZF) and zona reticularis (ZR) showed a notable hypertrophy in aged rats, which was due to rises in both the average volume and number of their parenchymal cells. The hypertrophy of ZF and ZR cells was in turn associated with increase in the volume of the mitochondrial compartment and proliferation of smooth endoplasmic reticulum, i.e., the two organelles involved in steroid-hormone synthesis. All these morphologic changes, conceivably due to the chronic exposure to high levels of circulating ACTH, are interpreted as a response enabling ZF and ZR to compensate for their age-dependent lowering in glucocorticoid secretion. Stereology also demonstrated that ZF and ZR cells underwent a striking age-related lipid-droplet repletion. Lipid droplets are the intracellular stores of cholesterol esters, the obligate precursors of steroid hormones in rats. This finding is in keeping with the contention that the mechanism underlying the age-dependent decline in rat-adrenal glucocorticoid secretion mainly involves impairments of the utilization of intracellular cholesterol previous to its intramitochondrial transformation to pregnenolone.

The effects of a severe streptozotocin (STZ)-induced diabetes on the morphology and function of the adrenal zona fasciculata were examined in rats with intact or pharmacologically interrupted hypothalamic-hypophyseal-adrenal axis. In animals with an intact hypothalamic-hypophyseal axis, STZ-diabetes induced hypertrophy of the cells of the zona fasciculata and a rise in the plasma corticosterone concentration. Conversely, in rats in which the hypothalamic-hypophyseal axis had been interrupted, experimental diabetes provoked atrophy of the zona fasciculata cells, and a lowering in the plasma corticosterone level. The effects of STZ-diabetes were completely reversed by insulin infusion in both groups of rats. The hypothesis is discussed that the chronic lack of insulin may directly inhibit the growth and steroidogenic capacity of the rat zona fasciculata and that this effect of experimental diabetes may be masked in rats with an intact hypothalamic-hypophyseal axis by the concurrent enhancement of ACTH release due to chronic stress resulting from the metabolic consequences of prolonged diabetes.

Endothelin-1 (ET-1), a potent 21 aminoacid residue vasoconstrictor peptide, is expressed in the human and rat adrenal cortex together with its receptor subtypes A and B (ETA and ETB), and stimulates steroids secretion. Although ET-1[1-21] is generated from big-ET-1 by an endothelin converting enzyme (ECE)-1, recent evidence indicates the existence of an alternative chymase-mediated biosynthetic pathway leading to the production of an ET-1[1-31] peptide. Since the role of the latter in adrenal pathophysiology is largely unknown, we investigated a) whether chymase is expressed at the gene and protein level in the human adrenal cortex; b) the secretagogue effect of ET-1[1-31], as compared to that of ET-1[1-21] on human adrenocortical cells. We found both ET-1[1-21] and ET-1[1-31] peptide concentration-dependently elicited a clear-cut secretory response by dispersed human adrenocortical cells, ET-1[1-31] being significantly less potent than ET-1[1-21]. The secretagogue effect of ET-1[1-31] was unaffected by high concentrations of phosphoramidone and thus did not require its conversion to ET-1[1-21] by ECE-1. The secretory response of adrenocortical cells to ET-1[1-31] was abolished by the ETA receptor antagonist BQ-123 and unaffected by the ETB receptor antagonist BQ-788. Since in humans the adrenocortical secretory response to ET-1[1-21] seems to occur via both ETA and ETB receptors, the weaker secretagogue action of ET-1[1-31] may be ascribed to a selective activation of ETA receptors. This contention is supported by findings that 1) that ET-1[1-31] displaced [125I]ET-1[1-21] autoradiographic binding to ETA, but not ETB receptors; and 2) that ET-1[1-31] was more effective than ET-1[1-21] in stimulating ETA-mediated cell proliferation. Collectively, these results raise the possibility that in human adrenals alternative cleavage of big-ET-1 by ECE-1 or chymase may lead to the production of either ET-1[1-21] mainly exerting secretagogue effects via both ETA and ETB receptors, or ET-1[1-31], mainly promoting growth via ETA receptors.

The aim of this study was to gain insight into the effects of 4-ammopyrazolo(3,4-d)pyrimidine (4-APP), a hypocholesterolemic drug, on the adrenal cortex of the hamster, representing an animal species in which steroidogenesis primarily relies on utilization of cholesterol synthesized de novo in the gland. 4-APP administration (1.5 mg/animal/day for 3 days) to intact or dexamethasone-suppressed hamsters resulted in a marked proliferation of adrenocortical cells. However, the volume of parenchymal cells was unchanged in intact animals and lowered in the zona glomerulosa (ZG) and zona reticularis (ZR) of dexamethasone-administered hamsters. In both groups of animals, 4-APP strikingly increased the volume of the lipid-droplet compartment and markedly reduced the surface area of smooth endoplasmic reticulum in ZF cells, without significantly affecting the volume of the mitochondrial compartment and the surface area of mitochondrial cristae. These morphologic changes displayed no evident correlation with adrenal cortisol content and secretion. Since most of the 4-APP-induced changes were not prevented by dexamethasone, it seems legitimate to suggest that they could mainly depend on a direct effect of 4-APP on the hamster adrenocortical cells.