Trophic and steroidogenic effects of water deprivation on the adrenal gland of the adult female rat

The effects of a 3-day water deprivation were studied in adult female rats in order to know what are the different zones of the adrenal gland and the hormonal factors involved in the growth and the activity of the adrenal gland. Water deprivation significantly increased plasma renin activity (PRA), plasma Angiotensin II (AII), vasopressin (AVP), epinephrine, aldosterone and corticosterone concentrations but did not modify the plasma adrenocorticotropin hormone (ACTH) level. Water deprivation significantly increased the absolute weight of the adrenal capsule containing the zona glomerulosa without modification of the density of cells per area unit suggesting that the growth of the adrenal capsule was due to a cell hyperplasia of the zona glomerulosa. Water deprivation significantly increased the density of AII type 1 (AT₁) receptors in the adrenal capsule but did not modify the density of AII type 2 (AT₂) receptors in the adrenal capsule and core containing the zona fasciculata, the zona reticularis and the medulla. The treatment of dehydrated female rats with captopril, which inhibits the angiotensin converting enzyme (ACE) in order to block the production of AII, significantly decreased the absolute weight of the adrenal capsule, plasma aldosterone and the density of AT₁ receptors in the adrenal capsule. The concentration of corticosterone in the plasma, the density of AT₂ receptors and the density of cells per unit area in the zona glomerulosa of the adrenal capsule were not affected by captopril-treatment. In conclusion, these results suggest that AII seems to be the main factor involved in the stimulation of the growth and the secretion of aldosterone by the adrenal capsule containing the zona glomerulosa during water deprivation. The low level of plasma ACTH is not involved in the growth of the adrenal gland but is probably responsible for the secretion of corticosterone by the zona fasciculata.     

The undifferentiated cell zone is a stem cell zone in adult rat adrenal cortex

The adrenal cortex of mammals has been known to consist of three morphologically and functionally distinct zones, i.e. the zona glomerulosa (zG), the zona fasciculata (zF) and the zona reticularis (zR), each of which secretes a specific corticosteroid different from those produced by the other two zones. We found previously, however, that an additional zone existed between zG and zF of adult rat adrenal cortex and that the cells in that zone were in a functionally undifferentiated state as an adrenocortical cell [Endocrinology 135, (1994) 431]: they were incapable of synthesizing highly active forms of corticosteroids, such as aldosterone and corticosterone, although they could produce their precursors. Hence, we named the zone as the undifferentiated cell zone (zU) of the adrenal cortex. Here we show that zU and its surroundings, i.e. the innermost portion of zG and the outermost portion of zF are the sites for cell replication in adult rat adrenal cortex and that the cells raised there migrate to other regions. Such cell replications in this region occur regardless of physiological conditions, such as the rise and fall of hormonal stimuli and circadian fluctuation of adrenocortical activities. On the bases of these and other findings previously described, we propose that zU is the stem cell zone of the adult rat adrenal cortex. Our recent success in isolating novel cell lines, which display an undifferentiated phenotype similar to that of zU cells, could facilitate the exploration of molecular mechanisms for the differentiation and development of the adrenocortical cells.     

Inhibitory effect of somatostatin on the growth and steroidogenic capacity of rat adrenal zona glomerulosa

Chronic administration of somatostatin induced atrophy of zona glomerulosa cells of rat adrenals and lowering of plasma aldosterone concentration. Zona fasciculata cells did not display any significant change and corticosterone plasma concentration was not significantly affected. These findings are interpreted to indicate that somatostatin exerts a direct inhibitory effect on the growth and steroidogenic capacity of rat adrenal zona glomerulosa.     

Secretion of aldosterone and corticosterone by the adrenals of Brattleboro rats in response to ACTH administration

The secretions of aldosterone and corticosterone in response to administration of 0,5 mUI of (1,24) ACTH (synacthène-Ciba) were measured in the adrenal venous blood of 15 Brattleboro female rats genetically lacking vasopressin and in 15 Long-Evans female rats, pretreated with dexamethasone. The secretions of aldosterone and corticosterone increased according to a similar profile in the two groups of animals: maximum values were 20-30 min. after ACTH injection; however the steroidogenic secretion of the adrenal cortex was always about 50% less in the Brattleboro female rats than in Long-Evans female rats. This result suggests mainly that vasopressin may be involved in the mechanisms which control the in vivo production of aldosterone by the adrenal glomerulosa cells.     

Effect of somatostatin on the zona glomerulosa of rats treated with angiotensin II or captopril: stereology and plasma hormone concentrations

Chronic somatostatin (SRIF) administration induced atrophy of zona glomerulosa cells of the rat adrenal cortex and a noticeable fall in the plasma concentration of aldosterone. The effects of SRIF were comparable with those of captopril, a specific inhibitor of the angiotensin-converting enzyme. SRIF completely abrogated the adrenoglomerulotrophic effects of angiotensin II (AII); the inhibitory actions of SRIF and captopril were not additive. The slight but significant enhancement of zona fasciculata cell growth and plasma corticosterone levels caused by chronic AII administration were ot reversed by SRIF. We interpret these data to indicate that SRIF specifically modulates the stimulatory effects of AII on the growth and steroidogenic capacity of rat zona glomerulosa.     

Serotoninergic stimulation of aldosterone secretion in vivo: role of the hypothalamo-pituitary adrenal axis.

The control of aldosterone secretion in vivo by serotonin was studied in conscious rats. Serial blood samples were taken from indwelling arterial cannulae before and after i.p. administration of 1 ml (4 g/l) 5-hydroxytryptophan (5-HTP), the precursor of serotonin (5-HT), or saline, and analysed for 5-HTP, serotonin, 5-hydroxyindoleacetic acid, plasma renin activity (PRA), corticosterone, aldosterone, sodium and potassium concentration. The relative contribution of the hypothalamo-pituitary adrenal axis was investigated in animals pretreated with the synthetic glucocorticoid dexamethasone. 5-HTP caused a significant increase in all parameters within 45 min except for plasma sodium and potassium. Saline administration showed no significant effect. Dexamethasone pretreatment significantly impaired the corticosterone and aldosterone response to 5-HTP, although the aldosterone response was merely attenuated. No other parameter was affected by dexamethasone pretreatment. The results show that administration of 5-HTP, which increases serum serotonin levels, stimulates PRA, corticosterone and aldosterone secretion. Dexamethasone pretreatment inhibits the aldosterone response, though not completely, suggesting that the stimulatory action of 5-HTP involves the release of ACTH, which stimulates corticosterone and aldosterone secretion by the adrenal cortex. The failure of dexamethasone to block the aldosterone response completely, suggests the involvement of other mechanisms such as the renin-angiotensin system or a direct action of serotonin on the adrenal zona glomerulosa.     

Molecular identity and gene expression of aldosterone synthase cytochrome P450

11Beta-hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11beta-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolated from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression.     

Induction of 5-aminolevulinate synthase by activators of steroid biosynthesis

Different cytochromes P450 are involved in steroid biosynthesis. These cytochromes have heme as the prosthetic group. We previously reported that ACTH, an activator of glucocorticoid biosynthesis in adrenal, requires heme biosynthesis for a maximal response. In the present study, we investigated the effect of ACTH, and the effect of two activators of the adrenal mineralocorticoid synthesis, endothelin-1 and low sodium diet on 5-aminolevulinate-synthase (ALA-s) mRNA. ALA-s is the rate-limiting enzyme in heme biosynthesis. It was found that infusion of rats with ACTH for 1 h caused an increase of adrenal ALA-s mRNA and activity accompanied by an increase in plasma corticosterone. CYP21, a cytochrome involved in the synthesis of both corticosterone and aldosterone, was not modified at the RNA level in adrenal glands by 1 h of ACTH infusion. Consistently, infusion of endothelin-1 for 1 h increased ALA-s mRNA and aldosterone content in adrenal gland without modifying CYP21 mRNA levels. To study if ALA-s is also regulated by the main physiological stimuli that increase adrenal mineralocorticoid secretion, we fed rats with low salt diet for 2 or 15 days. Low salt diet treatment increased adrenal gland ALA-s mRNA levels. On the other hand, the rapid stimulation of ALA-s mRNA by ACTH which acts through cyclic AMP was confirmed in H295R human adrenocortical cells, the only human adrenal cell line that has a steroid secretion pattern and regulation similar to primary cultures of adrenal cells. Our findings suggest that the acute activation of adrenal steroidogenic cytochromes by trophic hormones involves an increase in heme biosynthesis which will favor the production of active cytochromes.     

A possible association between aldosterone response to vasopressin and circadian change of aldosterone in the patients with aldosterone-producing adenoma

Vasopressin was reported to stimulate secretion of both cortisol and aldosterone through eutopic V1a receptors in adrenal gland. Recently, adrenal hyper-responsiveness of plasma cortisol to vasopressin with eutopic overexpession of V1a receptors has been reported in Cushing's syndrome, such as a majority of cases of ACTH-independent macronodular adrenal hyperplasia and some cases of Cushing's adenomas. There were a few reports regarding the aldosterone response to vasopressin in aldosterone-producing adenoma. The aim of our study was to investigate the aldosterone response to vasopressin and its pathophysiological roles in the patients with aldosterone-producing adenoma. Vasopressin-loading test was performed in 10 patients with aldosterone-producing adenoma, and in 16 patients with non-functioning adrenal tumors. The roles of the aldosterone response to vasopressin were analyzed in terms of hormonal secretion and the expression of V1a receptor mRNA on the operated adrenal gland in aldosterone-producing adenoma. We found that (1) a varying aldosterone response to vasopressin was observed, (2) absolute response of plasma aldosterone in aldosterone-producing adenoma was significantly higher than that in non-functioning tumor, (3) aldosterone response rate to vasopressin was significantly and negatively correlated with the decline rate (%) in plasma aldosterone from morning to evening in aldosterone-producing adenoma, (4) V1a receptor mRNA was expressed at various values in aldosterone-producing adenoma, and (5) surgical removal of aldosterone-producing adenoma eliminated the aldosterone response to vasopressin observed in patients with aldosterone-producing adenoma. These findings indicated that vasopressin might be involved in the coordination of aldosterone secretion through eutopic expression of V1a receptor in aldosterone-producing adenoma.     

Double NF1 Inactivation Affects Adrenocortical Function in NF1Prx1 Mice and a Human Patient

Background

Neurofibromatosis type I (NF1, MIM#162200) is a relatively frequent genetic condition, which predisposes to tumor formation. Apart from tumors, individuals with NF1 often exhibit endocrine abnormalities such as precocious puberty (2,5–5% of NF1 patients) and some cases of hypertension (16% of NF1 patients). Several cases of adrenal cortex adenomas have been described in NF1 individuals supporting the notion that neurofibromin might play a role in adrenal cortex homeostasis. However, no experimental data were available to prove this hypothesis.

Materials and Methods

We analysed Nf1Prx1 mice and one case of adrenal cortical hyperplasia in a NF1patient.

Results

In Nf1Prx1 mice Nf1 is inactivated in the developing limbs, head mesenchyme as well as in the adrenal gland cortex, but not the adrenal medulla or brain. We show that adrenal gland size is increased in NF1Prx1 mice. Nf1Prx1 female mice showed corticosterone and aldosterone overproduction. Molecular analysis of Nf1 deficient adrenals revealed deregulation of multiple proteins, including steroidogenic acute regulatory protein (StAR), a vital mitochondrial factor promoting transfer of cholesterol into steroid making mitochondria. This was associated with a marked upregulation of MAPK pathway and a female specific increase of cAMP concentration in murine adrenal lysates. Complementarily, we characterized a patient with neurofibromatosis type I with macronodular adrenal hyperplasia with ACTH-independent cortisol overproduction. Comparison of normal control tissue- and adrenal hyperplasia- derived genomic DNA revealed loss of heterozygosity (LOH) of the wild type NF1 allele, showing that biallelic NF1 gene inactivation occurred in the hyperplastic adrenal gland.

Conclusions

Our data suggest that biallelic loss of Nf1 induces autonomous adrenal hyper-activity. We conclude that Nf1 is involved in the regulation of adrenal cortex function in mice and humans.     

Hypertrophy and altered activity of the adrenal cortex in Homer 1 knockout mice

Homer 1 gene products are involved in synaptic transmission and plasticity, and hence, distinct behavioral abnormalities, including anxiety- and depression-like behaviors, have been observed in Homer 1 knockout (KO) mice. Here we report that Homer 1 KO mice additionally exhibit a pronounced endocrine phenotype, displaying a profoundly increased adrenal gland weight and increased adrenal/body weight ratio. Histological examinations of Homer 1 deficient adrenal glands revealed an increased size of the adrenal cortex, especially the sizes of the zona fasciculata and zona glomerulosa. Moreover, the plasma corticosterone and aldosterone were higher in Homer 1 KO than wild-type (WT) mice while the plasma ACTH levels were not different between the genotypes. The in vivo ACTH test revealed that corticosterone and aldosterone plasma levels were higher in saline injected Homer 1 KO mice than in WT mice (saline injected mice served as controls for the respective groups of ACTH-injected animals), but the magnitude of steroid responses to ACTH was similar in both genotypes. In contrast, an in vitro experiment performed on isolated cells of adrenal cortex clearly showed increased production of both steroids in response to ACTH in Homer 1 KO cells, which is in line with an ~8-fold increase in the expression of ACTH receptor mRNA in the adrenal cortex of these mutants. These results, together with the detection of Homer 1 mRNA and protein in the adrenal cortex of WT mice, indicate that Homer 1 directly affects the steroidogenic function of the adrenal glands.     

Endocrine-disrupting effects of nonylphenol in the newt, Triturus carnifex (Amphibia, Urodela)

The aim of our study was to verify whether environmental concentrations of nonylphenol influenced the adrenal gland of Triturus carnifex. Newts were exposed to 19 μg/L nominal concentration of nonylphenol throughout the periods of December-January and March-April, corresponding to different stages of the chromaffin cell functional cycle. The morphological features of the steroidogenic and chromaffin tissues, and the serum levels of ACTH, aldosterone, corticosterone, norepinephrine and epinephrine were evaluated. Nonylphenol did not influence ACTH serum levels. During the two periods examined, the steroidogenic tissue had the same reaction: the quantity of cytoplasmic lipids, and the corticosteroid serum levels, decreased, suggesting the inhibition of synthesis and release of corticosteroids. During the two periods examined, the chromaffin tissue reacted differently to nonylphenol. During December-January, the numeric ratio of norepinephrine granules to epinephrine granules, and the epinephrine serum levels, increased, suggesting the stimulation of epinephrine release. During March-April, the numeric ratio of norepinephrine granules to epinephrine granules did not change, and the norepinephrine serum levels decreased, suggesting the inhibition of norepinephrine release. Our results show that nonylphenol influences the activity of the newt adrenal gland; considering the physiological role of this gland, our results suggest that nonylphenol may contribute to amphibian decline.     

Functional significance of interrenal cell zonation in the adrenal gland of the duck (Anas platyrhynchos)

Slices of whole adrenal gland tissue, incubated in vitro in the presence of ACTH for 1 h and 2 h produced corticosterone and aldosterone in constant ratio (16:1). Tangential slices taken from the region immediately below the connective tissue capsule and slices taken from deeper regions of the gland consisted primarily of cells conforming to the distinct structural characteristics of the subcapsular zone (SCZ) and inner zone (IZ) tissues respectively. When samples were incubated in the presence of ACTH for 1 h and 2 h, the interrenal cells of the SCZ produced relatively more aldosterone than cells taken from the IZ of the gland. The corticosterone: aldosterone ratio for the IZ after 1 h (68:1) and after 2 h (102:1) were ten times greater than the ratios for the SCZ after 1 h (7:1) and after 2 h (10:1). The SCZ slices were not more than 60 cells thick and consisted of cells arranged in cords. These cells contained irregular nuclei, mitochondria with shelf-like cristae and a moderate abundance of smooth endoplasmic reticulum. In contrast, the production of large amounts of corticosterone by the cells of the IZ was associated with tissue containing more vascular space than the SCZ and the cells contained large round nuclei surrounded by an abundance of smooth endoplasmic reticulum and the mitochondria had tubular rather than shelf-like cristae.     

Effect of pentagastrin on steroid secretion and proliferative activity of regenerating rat adrenal cortex

The effects of three subcutaneous injections of 3 nmol/100 g body weight of the cholecystokinin type 2 (CCK2) receptor agonist pentagastrin on adrenocorticotrophic hormone (ACTH) and corticosterone secretion and proliferative activity of regenerating rat adrenal cortex were investigated. Pentagastrin did not alter either ACTH and corticosterone plasma concentrations or the adrenal mitotic index at day 5 of regeneration. In contrast, it increased (by about 50%) the adrenal mitotic index at day 8 of regeneration, and the effect was blocked by the simultaneous administration of equimolar doses of the CCK2-receptor antagonist PD-135,158. It is suggested that the activation of CCK2 receptors exerts a growth promoting action on the regenerating rat adrenal cortex.     

Sodium depletion of rats induces a mitochondrial protein in the zona glomerulosa of the adrenal cortex

Sodium restriction of rats resulted in a marked increase in the concentration of a mitochondrial protein with a mol. wt of 49000 in the zona glomerulosa of the adrenal cortex. The same protein had been previously induced in potassium deficient rats by potassium repletion. Since both sodium depletion and potassium repletion are known to stimulate the activity of the mitochondrial mixed function oxidase involved in the conversion of corticosterone to aldosterone, the present observation supports the hypothesis that the potassium-induced protein might represent cytochrome P-450CMO (corticosterone methyl oxidase).     

Evidence that an extrahypothalamic pituitary corticotropin-releasing hormone (CRH)/adrenocorticotropin (ACTH) system controls adrenal growth and secretion in rats

Within two weeks, hypophysectomy induced in rats a striking decrease in the level of circulating ACTH (the concentration of which was at the limit of sensitivity of our assay system), coupled with a net reduction in the plasma corticosterone concentration and an evident adrenal atrophy. Zona fasciculata, the main producer of glucocorticoids, was decreased in volume, due to a lowering in both the number and average volume of its parenchymal cells. Subcutaneous ACTH infusion (0.1 pmol·min^-1), administered during the last week following hypophysectomy, restored the normal blood level of ACTH and completely reversed all effects of hypophysectomy on the adrenals. Subcutaneous infusion for one week with -helical-CRH or corticotropin-inhibiting peptide (1 nmol·min^-1), which are competitive inhibitors of CRH and ACTH, evoked a further significant lowering of plasma corticosterone concentration and markedly enhanced adrenal atrophy in hypophysectomized rats. These findings strongly suggest that an extrahypothalamic pituitary CRH/ACTH system may be involved in the maintenance of the growth and steroidogenic secretory activity of the rat adrenal cortex.     

Cholesterol side-chain cleavage and 11 beta-hydroxylation are inhibited by gossypol in adrenal cortex mitochondria

Cholesterol side-chain cleavage and 11 beta-hydroxylation were assessed in isolated adrenal cortex mitochondria by formation of pregnenolone and corticosterone, respectively, in the presence and absence of gossypol. Pregnenolone biosynthesis was inhibited when increasing concentrations of gossypol were added. The control value of 4 nmol min-1 mg-1 dropped to 2 nmol min-1 mg-1 with 30 microM of the drug in the incubation medium. A more pronounced inhibitory effect was observed upon 11 beta-hydroxylation of steroids; I50 was 11 microM. Seventy-five percent of corticosterone production was impaired when 30 microM of gossypol were present. Bovine serum albumin prevented and reversed the inhibitory action of the drug. Kinetic studies showed a linear mixed type inhibition, suggesting a direct action of the drug upon the enzymatic complex. This study demonstrates a direct inhibitory effect of gossypol upon the steroidogenic enzymes located in the inner mitochondrial membrane of the adrenal cortex.     

Role of calcium in stimulus-secretion coupling on isolated frog interrenal gland

The influence of extracellular calcium concentration on the steroidogenic response to ACTH and to the angiotensin II analogue [Sar1-Val5]AII has been studied in the frog, using a perfusion system technique. The release of corticosterone and aldosterone in the effluent medium was measured by specific radioimmunoassays. In calcium-free medium the stimulatory effect of ACTH (10(-9) M) was completely abolished whereas the response to dbcAMP (5 mM) was unchanged indicating that the role of calcium takes place before the formation of cAMP. Conversely, in the absence of calcium, angiotensin II (10(-7) M) was still able to stimulate corticosterone and aldosterone production. Addition of Co2+ (4 mM), a calcium antagonist, to the perfusion medium, inhibited partially the response of adrenal tissue to ACTH, dbcAMP and angiotensin. The voltage-dependent calcium channel blocker verapamil (10(-6) induced a dose-related inhibition of the corticotropic effect of ACTH. At the higher dose (10(-4) M), verapamil totally inhibited the stimulation of corticosterone and aldosterone production induced by ACTH. By contrast, at the same dose it did not alter the stimulatory effect of forskolin (2.4 X 10(-7)M) on corticosterone output, but significantly diminished forskolin-induced aldosterone response. Similarly, angiotensin-stimulated corticosterone production was slightly inhibited by 10(-4) M verapamil, whereas aldosterone response to angiotensin was totally abolished, indicating that verapamil may act intracellularly to block the conversion of corticosterone to aldosterone. Taken together, these results indicate that, in amphibians extracellular calcium is essential for the action of ACTH, either for the binding of the hormone to its receptor and/or for the transduction of the information from hormone-receptor complex to the adenylate cyclase moiety and that the mechanism of action of angiotensin does not involve calcium uptake by adrenocortical cells.