Effect of ACTH on circadian periodicity of nuclear volume and mitotic division in the zona fasciculata externa of the adrenal cortex of mice.

In cells of the zona fasciculata externa of the adrenal cortex of mice, the maximal value of nuclear volume is observed in evening and night time, while the mitotic peak occurs in the early part of the day. Ten day subcutaneous injection of 1.5 units of ACTH twice in 24 hr produced nuclear hypertrophy and stimulation of mitotic activity of cells of the zona fasciculata externa. The circadian periodicity of nuclear volume in mice injected with ACTH is disturbed, while the circadian rhythm of mitotic activity is retained.     

Investigations on the effects of long-term administration of a methionine-enkephalin analogue on the adrenal zona fasciculata of rats treated with dexamethasone or dexamethasone and ACTH

Prolonged (7 days) methionine-enkephalin (DALA) treatment provoked a dose-dependent increase in the volume of zona fasciculata cells of dexamethasone-administered rats, along with a notable rise in the plasma concentration of corticosterone and the activity of 11 beta-hydroxylase. Comparable dose-dependent effects were observed after chronic administration of ACTH to dexamethasone-suppressed rats. The chronic administration of the maximum dose of DALA (500 micrograms/kg/day) was found to significantly further the trophic action of ACTH on the zona fasciculata of dexamethasone-treated animals. It is suggested that enkephalins act independently of and synergistically with ACTH in stimulating the growth and steroidogenic capacity of the rat adrenal zona fasciculata.     

Proliferation and distribution of adrenocortical cells in ACTH treated female hamsters

The aim of the study was the analysis, by means of combined stereologic methods and metaphase arrest technique of the adrenocortical growth of ACTH treated hamster. Adult female hamsters were treated daily with 50 micrograms ACTH (Synacthen Depot, Ciba) for 3, 6 and 9 days. Between days 3 and 9 nearly linear increase in adrenal gland weight was observed, mainly due to the enlargement of the fasciculata and reticularis zones. Throughout the experiment, there was no marked change in the number of the zona glomerulosa cells while from the day 3 a notable increase in the number of fasciculata and reticularis cells occurred. Prolonged ACTH treatment resulted in a significant increase in 3H-thymidine incorporation with the highest value on day 6 and subsequent drop on day 9. Also metaphase index of adrenocortical cells notably increased due to ACTH administration and again the highest value was found on day 6 of experiment. From stereologic data and from metaphase index, adrenocortical cell birth-rates in individual adrenocortical zones and in the entire cortex were calculated. There were great differences between the values calculated from metaphase index and those obtained from stereologic data, with the former significantly lower. ACTH enhances centripetal migration of parenchymal cells in the hamster adrenal cortex and causes their accumulation in the zona reticularis.     

Proliferation and distribution of adrenocortical cells in the gland of ACTH- or dexamethasone-treated rats

The effects of prolonged (7-day) ACTH and dexamethasone administrations on rat adrenocortical-cell turnover have been investigated by combined stereological and metaphase-arrest techniques. ACTH was found to increase the number of parenchymal cells in each adrenal zone; however, ACTH altered the cell distribution in the cortex, lowering their percentage in the zona glomerulosa (ZG) and zona fasciculata (ZF) and enhancing it in the zona reticularis (ZR). The cell birth-rate was markedly raised by ACTH exclusively in ZG and ZF. Dexamethasone notably decreased the number of ZF and ZR cells, without altering that of ZG cells. Moreover, dexamethasone increased the percentage of parenchymal cells in ZG and ZF, and lowered it in ZR. In the adrenal cortices of dexamethasone-administered animals, metaphases were virtually absent. These data indicate that ACTH increases the cell birthrate in ZG and possibly ZF, and enhances the centripetal migration of newly-formed cells and their accumulation in ZR. Dexamethasone inhibits both proliferation of adrenocortical cells in the outer cortical layers and their centripetal migration into ZR. Moreover, it appears to cause parenchymal-cell loss in the inner adrenocortical layers.     

Evidence that long-term methionine-enkephalin administration stimulates rat adrenal zona fasciculata

Short-term methionine-enkephalin (DALA) treatment did not induce evident changes in the adrenal zona fasciculata and in the basal corticosterone output of dexamethasone-treated rats administered with maintenance doses of ACTH. Conversely, prolonged (5 days) DALA treatment caused a notable hypertrophy of zona fasciculata cells, along with a significant increase in the plasma concentration of corticosterone. It is suggested that enkephalins exert a trophic action on the rat zona fasciculata.     

Immunocytochemical localization of ACTH-like immunoreactivity in rat adrenal glomerulosa and fasciculata cells

The role of ACTH in the synthesis of the adrenocortical hormones has been largely described. In order to investigate the localization of this peptide at the subcellular level of the adrenal glomerulosa and fasciculata cells, an immunocytological method was used. Rat adrenals were fixed and frozen. Ultrathin sections obtained by cryoultramicrotomy, were incubated with anti-beta (1-24) ACTH or anti-alpha (17-39) ACTH sera. The antigen-antibody reaction was detected by PAP complexes (revealed by 4-chloro-1-naphthol) or with protein A-colloidal gold or IgG-colloidal gold. The results obtained were the same whatever the antisera of the technique employed. All the cells of the adrenal zona glomerulosa and zona fasciculata were labelled. ACTH-like immunoreactivity in zona glomerulosa and zona fasciculata cells was observed at the plasma membrane level, in cytoplasmic matrix, mitochondria and nucleus (in the euchromatin close to the heterochromatin aggregations and, occasionally, associated with the nucleolus). No immunoreactivity was observed when non-immune serum or anti-ACTH serum preincubated with ACTH were used, nor there was any modification of the immunocytochemical reaction when anti-ACTH serum incubated with heterologous antigens was employed. These data, demonstrate the presence of endogenous ACTH in both adrenal glomerulosa and fasciculata cells, and suggest that the peptide is internalized after binding to the plasma membrane.     

Developmental aspects of the hypothalamic-pituitary-adrenal axis

The ovine fetal adrenal cortex and pituitary are functional secretory organs by the end of the first third of gestation (term is 142-152 days). By half-way through gestation the zona glomerulosa is mature morphologically, more than 80% of the aldosterone in fetal blood is of fetal adrenal origin, but conventional stimuli, for example, increased plasma K+ or angiotensin II, do not increase aldosterone secretion until near term. The zona fasciculata is immature histologically, relatively unresponsive to ACTH, and contributes less than 10% of the cortisol in fetal blood between 100 and 120 days of gestation. After this time the zona fasciculata cells begin to mature, to respond to ACTH and to produce an increasing proportion of the cortisol in fetal blood. A functional relationship between hypothalamus-pituitary-adrenal cortex matures over the last fifth of gestation. It is hypothesized that cortisol exerts a local effect in maturation of fetal zona fasciculata cells, such that low concentrations of ACTH have increasingly larger effects on growth and secretion of the fasciculata and that the level of negative feedback by cortisol on the hypothalamic-pituitary axis is reset. The analogy is drawn between the changes in gonadotrophin and gonadal hormones which culminates in puberty in man and the changes in ACTH and cortisol which culminate in parturition in sheep.     

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.     

The relationship between adrenal vascular events and steroid secretion: the role of mast cells and endothelin.

The actions of ACTH on the adrenal cortex are known to be 2-fold. In addition to increased steroidogenesis, ACTH also causes marked vasodilation, reflected by an increased rate of blood flow through the gland. Our studies, using the in situ isolated perfused rat adrenal preparation, have shown that zona fasciculata function and corticosterone secretion are closely related to vascular events, with an increase in perfusion medium flow rate causing an increase in corticosterone secretion, in the absence of any known stimulant. These observations give rise to two important questions: how does ACTH stimulate blood flow; and how does increased blood (or perfusion medium) flow stimulate steroidogenesis? Addressing the first question, we have recently identified mast cells in the adrenal capsule, and shown that Compound 48/80, a mast cell degranulator, mimics the actions of ACTH on adrenal blood flow and corticosterone secretion. We have also demonstrated an inhibition of the adrenal vascular response to ACTH in the presence of disodium cromoglycate, which prevents mast cell degranulation. We conclude, therefore, that ACTH stimulates adrenal blood flow by its actions on mast cells in the adrenal capsule. Addressing the second question, we looked at the role of endothelin in the rat adrenal cortex. Endothelin 1, 2 and 3 caused significant stimulation of steroid secretion by collagenase dispersed cells from both the zona glomerulosa and the zona fasciculata. A sensitive response was seen, with significant stimulation at an endothelin concentration of 10(-13) mol/l or lower. Endothelin secretion by the in situ isolated perfused rat adrenal gland was measured using the Amersham assay kit. Administration of ACTH (300 fmol) caused an increase in the rate of immunoreactive endothelin secretion, from an average of 28.7 +/- 2.6 to 52.6 +/- 6 fmol/10 min (P less than 0.01, n = 5). An increase in immunoreactive endothelin secretion was also seen in response to histamine, an adrenal vasodilator, which stimulates corticosterone secretion in the intact gland, but has no effect on collagenase-dispersed cells. From these data we conclude that endothelin may mediate the effects of vasodilation on corticosterone secretion, and this mechanism may explain some of the differences in response characteristics between the intact gland and dispersed cells.     

促肾上腺皮质激素引起大鼠肾上腺c-fos原癌基因表达的免疫组化研究

采用冰冻切片及免疫组化法观察了注射促肾上腺皮质激素(ACTH,75U/kg)、胰岛素及正常对照大鼠中肾上腺各部分c-fos原癌基因表达产物Fos蛋白的出现和分布特点。结果表明注射ACTH后90min,大鼠肾上腺皮质网状带出现Fos蛋白染色阳性细胞,阳性染色物集中于细胞核,肾上腺皮质束状带仅见少数Fos蛋白染色阳性细胞,肾上腺髓质则未见Fos蛋白染色阳性细胞。与注射ACTH相反,注射胰岛素引起肾上腺髓质出现Fos蛋白染色阳性细胞。注射生理盐水对照组动物肾上腺皮质和髓质均未见Fos蛋白染色阳性细胞。上述结果表明,注射ACTH或胰岛素可以引起大鼠肾上腺不同部位c-fos原癌基因表达。     

The subcapsular blastema of the adrenal cortex of the swine after continuous long-term infusion of exogenous ACTH]

After long time application of homologous ACTH the morphokinesis of the adrenal cortex of the pig was investigated experimentally. Following results were obtained: 1. In view of the controls the absolute and relative weight of the adrenals is raised considerably. 2. The progressive transformation is followed by the disappearance of the zonal structure of the adrenal cortex, and the parenchyma get the picture of fasciculata cells generally. 3. Nearly exclusive the zona fasciculata consists of great, pale activated spongiocytes with 2 nucleoli frequently. Topochemically glycogen and the lipids are inconstant, however the histochemical activity of succinodehydrogenase, lactate dehydrogenase, acid and alkaline phosphatase are considerable raised in regard of the controls. 4. The zona fasciculata contains degenerated cells isolated only. Signs of extensive regressive changes are not present. 5. The zona glomerulosa is dissolving or eliminating respectively. The consequences for the synthesis of the adrenal steroid hormones are discussed. 6. A large, spongy subcapsular blastema with several cell layers and a rich capillary network develop between the fibrous capsula of the adrenal and the zona fasiculata. The fasciculata cells are the direct continuation of the subcapsular blastema. The blastema contains neither glycogen nor lipids and histochemical activities of the enzymes are absent, too. The significance of the subcapsular blastema for the morphological and functional adaptation of the adrenal cortex in stress are discussed. Under the conditions of the closed hypothalamo-hypophyseal-adrenal control system the new origin of cells (hyperplasia) is not significant for the morphokinetic adaptive reactions of the adrenal cortex. Rather the subcapsular blastema represents a reserve area which after the destruction of the endocrine parenchyma through specific pathogens the organism enabled to the regeneration of the adrenal cortex.     

The influence of castration on cell proliferation in the adrenal cortex of female rats

Proliferation kinetics in the zones of the adrenal cortex of female rats after castration are reported. Following ovariectomy a wave of increased cell proliferation was observed in the zona glomerulosa and zona fasciculata after 1 and 2 days respectively. Increased cell proliferation in the zona reticularis was only slight. Cell proliferation returned to the level in control animals at 4 weeks. It is assumed that the regulative process following ovariectomy takes place directly in the adrenal cortex but it is possible that steroid metabolism in the liver is of importance. No relationship between the 3H-TdR labelling index (LI) and the mitotic index (MI) was observed.     

Chronic stress induces adrenal hyperplasia and hypertrophy in a subregion-specific manner

The adrenal gland is an essential stress-responsive organ that is part of both the hypothalamic-pituitary-adrenal axis and the sympatho-adrenomedullary system. Chronic stress exposure commonly increases adrenal weight, but it is not known to what extent this growth is due to cellular hyperplasia or hypertrophy and whether it is subregion specific. Moreover, it is not clear whether increased production of adrenal glucocorticoid after chronic stress is due to increased sensitivity to adrenocorticotropic hormone (ACTH) vs. increased maximal output. The present studies use a 14-day chronic variable stress (CVS) paradigm in adult male rats to assess the effects of chronic stress on adrenal growth and corticosterone steroidogenesis. Exogenous ACTH administration (0-895 ng/100 g body wt) to dexamethasone-blocked rats demonstrated that CVS increased maximal plasma and adrenal corticosterone responses to ACTH without affecting sensitivity. This enhanced function was associated with increased adrenal weight, DNA and RNA content, and RNA/DNA ratio after CVS, suggesting that both cellular hyperplasia and hypertrophy occurred. Unbiased stereological counting of cells labeled for Ki67 (cell division marker) or 4,6-diamidino-2-phenylindole (nuclear marker), combined with zone specific markers, showed that CVS induced hyperplasia in the outer zona fasciculata, hypertrophy in the inner zona fasciculata and medulla, and reduced cell size in the zona glomerulosa. Collectively, these results demonstrate that increased adrenal weight after CVS is due to hyperplasia and hypertrophy that occur in specific adrenal subregions and is associated with increased maximal corticosterone responses to ACTH. These chronic stress-induced changes in adrenal growth and function may have implications for patients with stress-related disorders.     

In vitro studies of nuclear volume of rat adrenocortical cells: lace of ACTH effect

The effect of ACTH on nuclear volume of adrenocortical cells in the zona fasciculata of rat adrenal cortex was examined in vitro. Sections of adrenal gland were incubated for 60 or 90 min in Krebs-Ringer's solution with 1% glucose in the presence of ACTH, actinomycin D, cycloheximide and aminoglutethimide. ACTH, despite its clear effect in stimulating steroidogenesis, did not exert a direct effect on the nuclear volume of cells studied. This phenomenon is not dependent upon the stimulation of steroidogenesis, since aminoglutethimide does not influence the nuclear volume of adrenocortical cells studied; rather, ACTH in the presence of aminoglutethimide leads to a decrease in their volume. Actinomycin D does not influence nuclear volume while after incubation with cycloheximide nuclei were larger than the control. The presence of ACTH did not alter this effect. These results indicate no relationship between the degree of corticosterone output and nuclear volume in rat adrenocortical cells of the zona fasciculata in vitro.     

Effect of long-term inhibition of hydroxy-methylglutaryl coenzyme A reductase by mevinolin on the zona fasciculata of rat adrenal cortex. A combined morphometric and biochemical study

The effects of a 7-day infusion with mevinolin, a potent competitive inhibitor of hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase, on the adrenal zona fasciculata were examined in normal and dexamethasone/ACTH-treated rats. In both groups of animals, the drug caused a lowering in plasma and intra-adrenal cholesterol concentrations, as well as a slight decrease in the blood level of corticosterone. Morphometry of zona fasciculata cells showed that specific mevinolin-induced changes (i.e. those occurring in both groups of rats and therefore not due to enhanced release of ACTH following decrease in circulating corticosterone) are severe lipid-droplet depletion and a conspicuous increase in smooth endoplasmic reticulum (SER) and peroxisomes. The hypothesis is discussed that these morphological changes express a compensatory response of zona fasciculata cells to counteract the mevinolin-induced inhibition of cholesterol synthesis in both liver and adrenal cortex.     

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.     

Synthetic modified N-POMC1–28 controls in vivo proliferation and blocks apoptosis in rat adrenal cortex

The identity of the pro-opiomelanocortin (POMC)-derived mitogen in the adrenal cortex has been historically controversial. We have used well-established in vivo models, viz., hypophysectomized (Hyp) or dexamethasone (Dex)-treated rats, to study the effect of the synthetic modified peptide N-terminal POMC (N-POMC_1–28) on DNA synthesis in the adrenal cortex, as assessed by BrdU incorporation and compared with adrenocorticotropic hormone (ACTH). We evaluated the importance of disulfide bridges on proliferation by employing N-POMC_1–28 without disulfide bridges and with methionines replacing cysteines. Acute administration of synthetic modified N-POMC_1–28 distinctly increased DNA synthesis in the zona glomerulosa and zona fasciculata, but not in the zona reticularis in Hyp rats, whereas in Dex-treated rats, this peptide was effective in all adrenal zones. ACTH administration led to an increase of BrdU-positive cells in all adrenal zones irrespective of the depletion of Hyp or Dex-POMC peptides. The use of the ACTH antagonist, ACTH_7–38, confirmed the direct participation of ACTH in proliferation. Two different approaches to measure apoptosis revealed that both peptides similarly exerted a protective effect on all adrenocortical zones, blocking the apoptotic cell death induced by hypophysectomy. Thus, ACTH_1–39 and N-POMC_1–28 have similar actions suggesting that the disulfide bridges are important but not essential. Both peptides seem to be important factors determining adrenocortical cell survival throughout the adrenal cortex, reinforcing the idea that each zone can be renewed from within itself.