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Inhibitory effects of a pineal extract on adrenal cortex. Lack of competition with ACTH.

The purpose of the present investigation was to study the mode of action of a crude aqueous pineal extract (CAPE) on corticosterone (B) production from ACTH-mediated isolated adrenal cortex cells. Corticosterone production from a heterogenous adrenal cortex cell population, isolated from 8 male Sprague-Dawley rats, was measured fluorimetrically. CAPE (25 microliters) was tested in this system using ACTH (0--5,000 pg/ml) and dibutyryl-c-AMP (0--100 nM/ml) as stimuli for a period of 1 h. In a separate experiment, CAPE (25 microliters) was administered to ACTH (50 pg/ml) stimulated adrenal cortex cells for 15, 30, 60, and 120 min incubation periods. CAPE significantly decreased B produced by adrenal cortex cells at all doses of ACTH administered. CAPE also decreased the B produced by adrenal cortex cells when dibutyryl-c-AMP was used as a stimulus. The inhibitory effect of CAPE was manifest at some point in time between 30 and 60 min. It was significant at 60 min and highly significant at 120 min. It is evident from these data that CAPE and ACTH are not competing for the same receptor site.

Adrenal Cortex↗

Zonal expression of dickkopf-3 and components of the Wnt signalling pathways in the human adrenal cortex.

The mechanisms underlying the differentiation of the adrenal cortex into zones are unclear. Microarray studies on RNA from microdissected zona reticularis (ZR) and zona fasciculata/zona glomerulosa (ZF/ZG) derived from adult human adrenal glands showed that a gene of the dickkopf family (DKK), DKK3, is differentially expressed in the zones. The Dickkopf proteins are morphogens involved in Wnt signalling. Northern blotting showed higher DKK3 transcript levels in ZF/ZG than ZR samples. In situ hybridization on adult human adrenal gland sections showed that DKK3 expression was much higher in the ZG than in the ZF or ZR. DKK3 expression was also higher in the medulla. We screened for expression of other members of the DKK family and the related Wingless-type mouse mammary tumor virus integration site gene family (WNT), frizzled (FZD), and dishevelled (DVL) gene families. Among dickkopf family members, only DKK3 was expressed at a detectable level in both human and mouse adrenocortical RNA samples. Consistent with previously published data on the effects of Wnt4 gene disruption in the mouse, we found only WNT4 expression within the WNT family in both human and mouse RNA. Northern blotting showed that WNT4 was expressed at a higher level in ZF/ZG cells than in ZR. The higher level of DKK3 and WNT4 expression in ZF/ZG cells was confirmed by real-time PCR. In the frizzled and dishevelled families we found FZD1, FZD2 and DVL3 transcripts in human adrenocortical RNA, and FZD2 and DVL3 in mouse adrenocortical RNA. These data show that a variety of genes of the Wnt signalling pathways are expressed in the adrenal cortex. The zonal distribution of DKK3 expression suggests that it could be involved in zonal differentiation or growth.

Adaptor Proteins, Signal Transducing↗

Bovine adrenal cortex adenylate cyclase: properties of the particulate enzyme and effects of guanyl nucleotides.

The preparation of a partially purified plasma membrane fraction from bovine adrenal cortex is described. Adenylate cyclase in this particulate preparation retained high sensitivity to ACTH and is also stimulated by 5'-guanylyl-imidodiphosphate [Gpp(NH)p]. GTP, in contrast to Gpp(NH)p, had very little intrinsic activity to stimulate activity to stimulate adenylate cyclase. GTP could however, with high affinity, inhibit the Gpp(NH)p effects on adenylate cyclase. When the concentration of creatine phosphate, a component of the ATP-regenerating system in the adenylate cyclase assay mixture, was lowered from 20 to 2 mM (at 0.1 mM ATP, 5 MM MG2+) GTP, dGTP and other nucleotides like ITP and much less UTP or CTP gained considerable intrinsic activity in the presence of ACTH to stimulate adenylate cyclase. The apparent affinities of the nucleotides for ACTH-stimulated adenylate cyclase from bovine adrenal cortex (at 2 mM creatine phosphate) were, GTP = dGTP greater than Gpp(NH)p greater than Gpp(CH2)p (5'-guanylyl-beta, gamma-methylene-diphosphonate) greater than ITP greater than UTP greater than CTP. These findings indicate that regulatory nucleotide binding sites exist for bovine adrenal cortex adenylate cyclase. Their specificity is similar to the nucleotide sites modulating angiotensin binding in bovine adrenal cortex plasma membranes (Glossmann et al., 1974a). The regulatory nucleotide binding sites for the adrenal cortex adenylate cyclase complex can also be identified under conditions where only Gpp(NH)p has high intrinsic activity (e.g. at 20 mM creatine phosphate) but other nucleotides like GTP act as antagonists. Both stimulants, ACTH and Gpp(NH)p, appear to remain firmly bound to the particulate membrane preparation, as suggested by preincubation experiments.

Adenosine Triphosphate↗

Ovine steroid 17alpha-hydroxylase cytochrome P450: characteristics of the hydroxylase and lyase activities of the adrenal cortex enzyme.

The steroid 17-hydroxylase cytochrome P450 (CYP17) found in mammalian adrenal and gonadal tissues typically exhibits not only steroid 17-hydroxylase activity but also C-17,20-lyase activity. These two reactions, catalyzed by CYP17, allow for the biosynthesis of the glucocorticoids in the adrenal cortex, as a result of the 17-hydroxylase activity, and for the biosynthesis of androgenic C(19) steroids in the adrenal cortex and gonads as a result of the additional lyase activity. A major difference between species with regard to adrenal steroidogenesis resides in the lyase activity of CYP17 toward the hydroxylated intermediates and in the fact that the secretion of C(19) steroids takes place, in some species, exclusively in the gonads. Ovine CYP17 expressed in HEK 293 cells converts progesterone to 17-hydroxyprogesterone and pregnenolone to dehydroepiandrosterone via 17-hydroxypregnenolone. In ovine adrenal microsomes, minimal if any lyase activity was observed toward either progesterone or pregnenolone. Others have demonstrated the involvement of cytochrome b(5) in the augmentation of CYP17 lyase activity. Although the presence of cytochrome b(5) in ovine adrenocortical microsomes was established, ovine adrenal microsomes did not convert pregnenolone or 17-hydroxypregnenolone to dehydroepiandrosterone. Furthermore the addition of purified ovine cytochrome b(5) to ovine adrenal microsomes did not promote lyase activity. We conclude that, in the ovine adrenal cortex, factors other than cytochrome b(5) influence the lyase activity of ovine CYP17.

17-alpha-Hydroxypregnenolone↗

Ectopic localisation of adrenal cortex.

We present a case of concurrence of ectopic adrenal cortex with a renal cell carcinoma. The diagnosis of the accessory adrenal tissue was made by CT-guided biopsy. With this case report, we draw attention to a specific differential diagnostic problem, policy and to the MR characteristics of ectopic adrenal cortex.

Adrenal Cortex↗

Differential production of adrenal steroids by purified cells of the human adrenal cortex is relative rather than absolute.

OBJECTIVES: The adrenal cortex produces aldosterone, cortisol and androgens in response to ACTH and angiotensin II. To define the differential response of morphologically distinct cells of the adrenal cortex, we examined the phenotypical and functional characteristics of human adrenocortical cells. RESULTS: Tumour growth factor-beta receptor-1 (TGFbeta-R1) and CYP-11 were found to be expressed predominantly in the zona fasciculata, whereas human leukocyte antigen (HLA-DR) and CYP-17 were localised to the zona reticularis. The angiotensin II receptor, AT-1, was found to be predominantly expressed in the zona glomerulosa. Adrenocortical cells, separated by density, yielded two distinct fractions which displayed differential growth patterns. Lipid-rich cells of fraction I expressed TGFbeta-R1 and produced significantly more cortisol relative to androstenedione than unseparated or fraction II cells, whereas lipid-poor cells of fraction II expressed HLA-DR and produced more androstenedione relative to cortisol in the presence of ACTH. Aldosterone production by fraction II was significantly greater than fraction I or unseparated cells. TGFbeta-R1-positive fasciculata-type cells separated into fraction I and HLA-DR-positive cells consistent with reticularis cells separated into fraction II. Aldosterone-producing cells indicative of glomerulosa cells separated into fraction II. CONCLUSIONS: Our findings are consistent with the concept that all adrenocortical cells are capable of producing a range of steroids, but the relative production of cortisol, androgen and aldosterone differs.

Adrenal Cortex↗

Adrenal cortex of fetal lamb: changes after hypophysectomy and effects of Synacthen on cytoarchitecture and secretory activity.

The effects of hypophysectomy on the cell population of the adrenal cortex has been examined with light and electron microscopy. The sensitivity of the adrenal cortical cells to exogenous ACTH in both normal and hypophysectomized fetuses has been investigated by comparing plasma cortisol concentrations with structural changes in the adrenal cortex. Hypophysectomy was carried out at about 100 d and the subsequent analysis was made at about 135 d on fetuses catheterized at least 6 d earlier. The zona fasciculata of intact fetuses contained about 25% mature and 75% immature cells at 130-136 d. After hypophysectomy this zone contained a uniform population of immature cells and no mature cells; there was a significant reduction in both adrenal weight and cortical thickness. The zona glomerulosa was unaffected by hypophysectomy. The infusion of Synacthen 0 . 25 mg/d I.V. for 48 h did not affect the zona glomerulosa but resulted in a 2-fold increase in the thickness of the zona fasciculata in hypophysectomized and intact fetuses. This cortical growth was due to both hypertrophy and hyperplasia. All the cells in the zona fasciculata were mature following Synacthen treatment in both groups. The most striking difference between the secretory response of the adrenal cortex to Synacthen in the two groups was found within 3-6 h of the onset of infusion. Within this period there was a significant increment in the plasma cortisol concentration in the intact fetuses while no response was observed in the hypophysectomized animals. After 12 h infusion by contrast, a steadily rising response was observed in both groups. There was a significant correlation between the final plasma cortisol concentration and both the adrenal weight and cortical thickness after 48 h treatment. The increased sensitivity of the adrenal cortex to exogenous ACTH as term approaches in the sheep is considered to be dependent upon the increasing proportion of mature zona fasciculata cells within the adrenal cortex.

Adrenal Cortex↗

Effect of adrenal medullectomy on the activity of COMT and MAO in adrenal cortex of control and stressed rats.

The effect of bilateral adrenal medullectomy on the activity of catecholamine degrading enzymes--catechol-o-methyl transferase (COMT) and monoamino oxidase (MAO)--in adrenal cortex and on the level of corticosterone in plasma was studied in the rats subjected either to a single (2.5 h) or repeated (2.5 h daily for 150 days omitting Saturdays and Sundays) immobilization stress. After medullectomy a significant increase of COMT activity was found in adrenal cortex of unstressed controls, while the activity of MAO was significantly decreased. Similar results were observed also in medullectomized animals subjected either to a single or repeated stress. The levels of corticosterone remained unchanged in medullectomized controls, but were significantly decreased in medullectomized and stressed animals. Although the mechanism still remains to be elucidated, these results may be considered as a further contribution to the understanding of the functional interrelations between the adrenal medulla and cortex.

Adrenal Cortex↗

Cell proliferation and renewal of mouse adrenal cortex.

Although many hypotheses concerning cell proliferation and renewal in the adrenal cortex of mammals have been proposed, this topic has so far not been elucidated. Adrenocortical cells of adult mammals have low proliferative activity and take a considerable length of time to be renewed. This makes it difficult to investigate the dynamic features of their proliferation. To clarify the cell kinetics, we undertook a long term study in mice using an autoradiographic technique. We radiolabelled almost all the cells throughout the body in newborn mice with the exception of the neurons in central nervous system by the frequent subcutaneous injections of [3H]thymidine every 6 h for 30 d (pulse labelling). After this sequence of pulse labelling, we observed autoradiographically a decrease in the number of 3H-labelled cells in the adrenal cortex as a result of replacement with proliferated unlabelled cells (renewed cells). Single injections of [3H]thymidine (flash labelling) was also performed to examine DNA synthesis in the adrenal cortex. The investigations indicated that the adrenocortical cells proliferate at the border between the zona glomerulosa and the zona fasciculata, and that renewed cells which proliferated in that region move with time bidirectionally towards the cortical surface and the inner (medullary) surface. Half of the cortical cells in the zona glomerulosa, zona fasciculata and zona reticularis were replaced by renewed cells in 30, 60 and 120 d respectively. It took 200 d for almost all cortical cells to be replaced by renewed cells.

Adrenal Cortex↗

Functional innervation of the adrenal cortex by the splanchnic nerve.

Secretion of steroid hormones by the adrenal cortex is required to maintain whole body homeostasis; that is the ability to maintain blood pressure and volume, carbohydrate, protein and fat metabolism and immune and nervous system function within normal limits is dependent on adrenocortical hormones. The premise of this report is that autonomic-endocrine interactions occurring in the adrenal cortex are required for normal control of steroid secretion. Under non-stress conditions when reduced steroid secretion is required, splanchnic neural activity appears to be inhibitory, whereas during stress conditions when elevated steroid secretion is necessary, neural activity is excitatory. The capacity for innervation to produce both inhibitory and excitatory effects suggests that neural input must be encoded differentially; encoding could be dependent on the neurotransmitter released or on the intra-adrenal target affected. Neural input could act directly at the adrenal cell to affect steroidogenesis or act indirectly by changing adrenal blood flow. An index of the role of innervation has been obtained by assessing adrenal corticosteroid secretion after splanchnicectomy, severing the thoracic splanchnic nerve which is the major source of innervation of the adrenal gland. This approach has resulted in alterations in corticosteroid secretion under non-stress and stress conditions, but in many cases has demonstrated no profound effect on in vivo steroidogenesis. It is likely that splanchnicectomy results in variable secretory responses in part due to the multiplicity of adrenal neurotransmitter systems that are regulated by the splanchnic nerve. Splanchnicectomy alters multiple neurotransmitters at different adrenal sites. Splanchnic innervation acts as an extra-ACTH mechanism in the control of adrenal corticosteroid secretion, yet further elucidation of the physiological conditions under which splanchnic neural activity affects function is clearly warranted.

Adrenal Cortex↗

Hormonal regulation of guanosine triphosphate cyclohydrolase activity and biopterin levels in the rat adrenal cortex.

The regulation of GTP-cyclohydrolase (GTP-CH) activity and tetrahydrobiopterin (BH4) levels in the adrenal cortex were studied in intact and hypophysectomized rats. Treatment with a single dose of reserpine (5 mg/kg) or insulin-induced hypoglycemia (4 h) elevated adrenocortical BH4 3-fold by 10 h; BH4 levels remained elevated after 24 h and returned to control levels by 48-72 h. GTP-CH was significantly increased 24 h after hypoglycemic shock, and the increased enzyme activity preceded the changes in BH4 levels after reserpine treatment. Two and a half hours of stress by immobilization also increased GTP-CH activity and BH4 levels in the adrenal cortex. The activities of sepiapterin reductase and dihydrofolate reductase, putative enzymes in the biosynthetic pathway from GTP to BH4, were not increased by reserpine. Both reserpine and insulin increased the apparent maximum velocity for GTP, with no increase in the affinity of the enzyme for its substrate, further suggesting that the experimental treatments induce the synthesis of GTP-CH. Hypophysectomy completely blocked the reserpine-dependent increase in both cortical GTP-CH activity and BH4 content. The administration of purified porcine ACTH to intact and hypophysectomized rats elevated adrenocortical GTP-CH activity and cofactor levels. Synthetic ACTH-(1-24) also enhanced the enzyme activity and BH4 levels in the adrenal cortex. Thus, pituitary control of adrenal cortical GTP-CH synthesis and biopterin levels appears to be mediated through the secretion of ACTH. The changes in enzyme activity and cofactor levels after activation of the hypothalamo-hypophyseal axis or ACTH administration suggest that BH4, a cofactor for certain monooxygenases, has some function, as yet unknown, in the adaptive changes of the adrenal cortex in response to stress.

Adrenal Cortex↗

Chicken ovalbumin upstream promoter transcription factor II in the human adrenal cortex and its disorders.

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an orphan member of the steroid/thyroid hormone receptor superfamily. COUP-TFII has been demonstrated to negatively regulate the transcriptional activity of adrenal 4-binding protein, a steroidogenic cell-specific transcription factor that activates the transcription of various steroidogenic P450 genes. We therefore examined immunolocalization of COUP-TFII in the human adrenal cortex and its disorders, including functioning and nonfunctioning cortical tumors, to study its possible correlation with adrenocortical steroidogenesis. In nonpathological adrenal cortex, COUP-TFII immunoreactivity was marked in the nuclei of adrenocortical cells in definitive and fetal zones from 16 gestational weeks to 2 months after birth. Immunoreactivity for COUP-TFII was marked in the zona glomerulosa and weak in the zonae fasciculata and reticularis from 7 months to 8 yr of age, but thereafter markedly decreased in these zones (P < 0.05, between age 7 months to 8 yr and 24-62 yr of age, respectively). In adrenocortical tumors, COUP-TFII immunoreactivity was marked in the nuclei of tumor cells of aldosteroma (H score, 134 +/- 15.9; P < 0.001 vs. Cushing's adenoma and P < 0.05 vs. nonfunctioning adenoma and carcinoma), modest in nonfunctioning adenoma (82.7 +/- 19.8) and adrenocortical carcinoma (79.6 +/- 56.3), and low in Cushing's adenoma (38.2 +/- 24.5). Results from immunoblotting performed in seven cases of adenomas were consistent with those of immunohistochemistry. In the attached nonneoplastic adrenal cortex of the adenomas, immunoreactivity for COUP-TFII was markedly increased compared to that in nonpathological adrenal cortex in adults and was especially marked in the zona glomerulosa in the attached adrenal of aldosteroma (P < 0.001) and the zona fasciculata in that of Cushing's adenoma (P < 0.05). COUP-TFII immunoreactivity was universally detected in stromal cells of the adrenal glands. These results suggest that COUP-TFII plays an important role in the regulation of steroidogenesis in human adrenal cortex and its disorders.

Adenoma↗

Proliferation of capsular stem cells induced by ACTH in the rat adrenal cortex.

Despite great efforts devoted to clarifying the localization of proliferative activity in the adrenal cortex, the agents that stimulate proliferation remain controversial, and the nature of the stem cells from which cortical cells differentiate is incompletely understood. We studied proliferative activity in the rat adrenal cortex using an immunohistochemical method to detect the presence of the Proliferating Cell Nuclear Antigen (PCNA) (an intranuclear enzyme whose synthesis reaches the maximum intensity during the S-phase of the cell cycle). Groups of six rats were subjected to daily intraperitoneal injection of either corticotropin (ACTH1-24--0.2 mg/kg), dexamethasone (Dexa--4 mg/kg) or 0.9% saline for three consecutive days and killed 24 h after the last injection. Adrenal weight was significantly increased by ACTH treatment and reduced by Dexa. Concentrations of endogenous ACTH in plasma were lower in the Dexa group than in controls, and curiously, this was true in the ACTH1-24 treated group as well, probably in consequence of the increased corticosterone levels providing negative feedback at the hypothalamic-pituitary level. Corticosterone levels, as expected, were increased by the ACTH stimulus and reduced by the use of Dexa. Proliferating cell nuclear antigen immunostaining was close to zero in Dexa treated animals and low in controls. In ACTH treated rats, a significantly increased number of cells were positively stained. Positive cells were identified in both in zona glomerulosa (ZG) and zona intermedia (ZI) but many were located in the capsule. Zona fasciculata (ZF) and zona reticularis (ZR) were devoid of staining in all of these cases. We conclude that pharmacological doses of ACTH induce proliferation of capsular fibroblasts. Following descriptions by early 20th century researchers it is possible that these cells may also be stem cells and differentiate into adrenal cortex cells.

Adrenal Cortex↗

[Mineralocorticoid function of the adrenal cortex and the renin--angiotensin system in Itsenko-Cushing's disease].

Mineralocorticoid function of the adrenal cortex and the activity of the renin-angiotensin system were studied in patients with Itsenko-Cushing's disease before (36 cases) and after (27 cases) the treatment with chloditan--an adrenocortical inhibitor. Minertalocorticoid function of the adrenal cortex was assessed by the blood immunoreactive aldosterone content, urinary 18-aldosterone-glucuronide excretion, and the rate of aldosterone secretion. Condition of the renin-angiotensin system was assesed by the blood renin activity. Aldosterone and renin concentration was studied by the radioimmunological method. There proved to be a reverse relationship between the blood aldosterone concentraiton and the severity of Itsenko-Cushing's disease. Elevation of the mineralocorticoid function of the adrenal cortex in Itsenko-Cushing's disease was due to activation of the renin angiotensin system. During clinical remission of the disease following a course of chloditan treatment the patient displayed an increased blood aldosterone level and a rise of its secretion. The minralocorticoid function of the adrenal cortex was intensified at the state of clinical remission because of an increased blood corticotropin concentration.

Adolescent↗

[Functional state of the adrenal cortex in patients with Parkinsonism before and after surgical treatment].

The results of biochemical examinations of the hormonal function of the adrenal cortex in 22 patients with Parkinson's disease are presented as seen prior and following stereotaxic operations on the ventro-lateral nucleus of the thalamus. It was found that parkinsonics have a decreased androgenic, glucocorticoid and mineralocorticoid function of the adrenal cortex. Long history and growing severity of the disease manifest themselves in a distinct inhibition of the adrenal cortex function. The importance of the changes in the functional state of the hypothalamo-hypophyseal system in the revealed disorders in the hormonal activity is emphasized. Along with a beneficial clinical effect surgery brings about an improvement of the functional state of the adrenal cortex.

17-Hydroxycorticosteroids↗

Focal lymphocytic infiltration in the adrenal cortex of the elderly: immunohistological analysis of infiltrating lymphocytes.

The incidence of mononuclear cell infiltration in the adrenal cortex was examined in autopsy cases of young and old subjects, and the infiltrating mononuclear cells were immunohistologically characterized by monoclonal antibodies. Histologically, 110 of 174 autopsy cases of persons greater than 60 years (63.2%) were shown to have mononuclear cell infiltration of varying degree within the adrenal cortex, whereas such a lesion was observed in lesser incidence (7.4%) in the 54 younger, control subjects aged less than 49 years. In addition, severely infiltrating lesions in the adrenal cortex were found frequently in the elderly greater than 70 years. Immunohistochemical study revealed that the infiltrating mononuclear cells were mainly composed of CD3+ T cells. The major proportion of CD3+ T cells expressed CD4, whereas CD8+ T cells were less in number. Moreover, a considerable proportion of CD4+ T cells was activated as judged by interleukin 2 receptor expression. These findings indicate that T lymphocytes infiltration in aged human adrenal cortex may represent a pre-clinical manifestation of organ-specific autoimmune adrenalitis which is based on autoimmunity associated with ageing process.

Adrenal Cortex↗