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J.-G. Lehoux and A. Lefebvre

ABSTRACT

Low-density lipoprotein (LDL) receptor mRNA was found in both rat and hamster adrenals. Within 30 min after ACTH administration a significant increase in the levels of both LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) mRNAs was observed in rat adrenals; these levels remained increased for up to 240 min. The increase in the levels of LDL receptor and HMG-CoA reductase mRNAs produced by ACTH was reduced by co-administration of aminoglutethimide while, at the same time, the adrenal cholesterol content of rats treated with both aminoglutethimide and ACTH was significantly increased compared with that in groups treated with ACTH alone. Cycloheximide also induced increased levels of rat adrenal mRNAs for LDL receptor and HMG-CoA reductase, but this effect was not additive with that of ACTH. These results suggest that, in the rat, the short-term effect of ACTH on the levels of mRNAs for the LDL receptor and HMG-CoA reductase is similarly controlled and might be mediated through changes in the adrenal cholesterol content. In the hamster adrenal, however, no significant fluctuations were found in the level of LDL receptor mRNA, although a marked increase was found in the level of HMG-CoA reductase mRNA, 2 h after ACTH administration. This indicates that an important effect of ACTH on cholesterol metabolism in the hamster adrenal is at the level of HMG-CoA reductase. In the hamster, therefore, where the main source of cholesterol for the adrenal gland is de-novo synthesis, it seems that a complex mechanism is involved in the control of LDL receptor gene expression.

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P. Enyedi, T. Balla, F. A. Antoni, and A. Spät

ABSTRACT

We have previously shown that arginine vasopressin (AVP) stimulates the production of aldosterone in isolated superfused adrenal glomerulosa cells by a mechanism that involves an increased turnover of phosphoinositides. In the present study we compared the characteristics of AVP- and angiotensin II (AII)-induced changes in phosphoinositide turnover and aldosterone production in the rat. Selected concentrations of the two peptides, which were equipotent in terms of the magnitude of changes induced in phosphoinositide turnover, stimulated aldosterone production to the same extent only in the initial phase of the stimulation. A sustained aldosterone response was only observed in AII-stimulated cells. On the other hand, the AVP-induced increase in incorporation of [32P]phosphate into phosphatidyl-inositol and the stimulation of inositol phosphate production were maintained during incubation. Preincubation of the cells with AVP failed to modify the effects of AII on phosphoinositide breakdown or aldosterone production. These results indicate that desensitization at the level of the receptor or at a post-receptor site is not responsible for the transient character of AVP-induced aldosterone production. Delayed activation of an inhibitory mechanism by AVP can also be excluded. Additivity of the stimulation of the phosphoinositide turnover observed at submaximally, but not maximally, effective concentrations of AII indicates that the two agonists act on the same phosphoinositide pool. We suggest that the sustained steroidogenic effect of AII involves an as yet unidentified mechanism, which is absent when the cells are stimulated with AVP.

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Philippe Huber, Christine Mallet, Elodie Faure, Christine Rampon, Marie-Hélène Prandini, Olivier Féraud, Stéphanie Bouillot, and Isabelle Vilgrain

Vascular endothelial-cadherin (VE-cadherin) is an endothelial cell-specific adhesion protein that is localised at cell–cell contacts. This molecule is an important determinant of vascular architecture and endothelial cell survival. In the adrenal cortex, steroidogenic and endothelial cells form a complex architecture. The adrenocorticotrophin hormone (ACTH) regulates gland homeostasis whose secretion is subjected to a negative feedback by adrenocorticosteroids. The aim of the present study was to determine whether VE-cadherin expression in the adrenal gland was regulated by hormonal challenge. We demonstrated that VE-cadherin protein levels were dramatically decreased (23.5 ± 3.7%) by dexamethasone injections in the mouse and were restored by ACTH within 7 days (94.9 ± 18.6%). Flow cytometry analysis of adrenal cells showed that the ratios of endothelial versus total adrenal cells were identical (35%) in dexamethasone- or ACTH-treated or untreated mice, suggesting that VE-cadherin expression could be regulated by ACTH. We demonstrate the existence of a transcriptional regulation of the VE-cadherin gene using transgenic mice carrying the chloramphenicol acetyl transferase gene under the control of the VE-cadherin promoter. Indeed, the promoter activity in the adrenals, but not in the lung or liver, was decreased in response to dexamethasone treatment (40 ± 1.3%) and was partially restored after gland regeneration by ACTH injection (82 ± 3%). In conclusion, our results show that transcription of a specific endothelial gene is controlled by the hypothalamo–pituitary axis and the data expand the knowledge regarding the role of ACTH in the regulation of the adrenal vascular network.

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E. Jimenez, S. Marsigliante, S. Barker, J. P. Hinson, and G. P. Vinson

ABSTRACT

Angiotensin II (AII) receptors were identified in rat tissue membranes by specific binding of 125I-labelled AII. Using an isoelectric focusing technique, two forms of the high-affinity AII receptor were identified in rat adrenal zona glomerulosa and liver membranes. These migrated to isoelectric points (pI) 6.8 and 6.7. Two low-affinity forms migrated to pI 6.5 and 6.3. The two high-affinity forms were in greatest abundance in the zona glomerulosa, while the low-affinity pI 6.5 isoform was predominant in liver membranes. In uterine membranes both low-affinity isoforms were observed, but there was only one of the high-affinity forms (pI 6.7).

Concentrations of AII receptor isoforms were increased in the zona glomerulosa of sodium-deprived rats.

Reduction of disulphide bridges with dithiothreitol (DTT) had different effects on the various AII receptor isoforms. Thus 1 mmol DTT/l caused a twofold increase in 125I-labelled AII binding in zona glomerulosa membranes. DTT produced no appreciable differences in specific AII binding in uterine membranes, whereas there was a 50% reduction of binding in liver membranes. At 20mmol/1, DTT greatly decreased AII binding in all tissues.

The data suggest the existence of multiple forms of AII receptors which may have different functions.

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A. N. Al-Othman, K. Docherty, M. W. Makgoba, M. C. Sheppard, and D. R. London

ABSTRACT

Congenital adrenal hyperplasia (CAH) is a family of inherited disorders of adrenal steroidogenesis, most commonly due to deficiency of P-450 21-hydroxylase (21-OH). There are two genes for 21-OH on the short arm of chromosome 6, the A gene which is thought to be inactive, and the B gene. These genes appear as 3·2 and 3·7 kb TaqI fragments on Southern blots. In a study of DNA from 60 normal controls with TaqI and a 21-OH cDNA probe, 12% exhibited a homozygous deletion of the A gene, and 22 and 8% heterozygous deletions of A and B genes respectively. TaqI analysis of eight patients with CAH revealed four without A or B gene deletions, three with heterozygous deletions of the B gene and one with a homozygous deletion of the B gene. On further analysis with KpnI, EcoRI, PvuII and BglII, however, these genotypes were amended to two with heterozygous deletions of the B gene and two with possible B to A gene conversions. The genotypes of the four patients without deletions remained unchanged.

RNA from CAH and Cushing's adrenal tissue was also analysed using A and B gene-specific oligodeoxynucleotide probes. B gene transcripts were detected in both CAH and Cushing's adrenals, while no A gene transcripts could be detected in either tissue. The level of B gene-derived mRNA was greater in the Cushing's adrenal than in the CAH adrenal, which in turn was greater than that in the adrenal from a normal individual.

These results suggest that there is a high frequency of 21-OH gene deletions in the normal population, but that TaqI alone is not capable of unequivocally identifying such deletions. The results also suggest that CAH is caused by heterogenous defects of the B gene. The defective gene, however, is transcriptionally active, indicating that the defect is not within the regulatory region.

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A Fleury, L Ducharme, and JG LeHoux

In this study, we report the cDNA cloning of hamster adrenal steroidogenic acute regulatory (StAR) protein and the effect of adrenocorticotrophin (ACTH) on its expression in vivo. A hamster adrenal cDNA library was screened using an 852 bp fragment obtained by polymerase chain reaction; this fragment corresponds to the entire coding sequence (CDS) of the hamster adrenal StAR cDNA. Ten clones of different lengths were isolated and sequenced. The longest clone was 1564 bp and contained 34 bp in the 5'-untranslated region, 852 bp in the CDS, and 678 bp in the 3'-untranslated region (3'-UTR). Two polyadenylation signal sequences were found in the 3'-UTR. The CDS of the ten isolated clones was identical, but six of these lacked the last 132 nucleotides in the 3'-UTR, thus indicating that they had used the first polyadenylation signal. The hamster StAR protein contains 284 amino acid residues, and is 91.9% homologous to mouse, 90.5% to rat, 86.4% to human, 85% to porcine, and 82.5% to bovine StAR protein. Southern blot analysis indicated the presence of only one StAR gene in the hamster genome. Northern blotting analysis revealed the presence of the StAR mRNA in male and female steroidogenic tissues, namely adrenals and gonads, but not in the liver or in the kidneys of either sex. Three mRNA species of 1.7, 3.1 and 5.3 kb were found in whole hamster adrenals. Administration of ACTH to hamsters provoked increases (two- to threefold) in the adrenal content of the StAR mRNA within 1 h in vivo. Western blotting analysis on adrenal mitochondria showed that the level of StAR protein was also significantly elevated (1.5-fold) 1 h after ACTH treatment.

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G. P. Vinson, S. M. Laird, J. P. Hinson, N. Mallick, S. Marsigliante, and R. Teja

ABSTRACT

When rat adrenal whole capsules, containing the zona glomerulosa, were incubated, addition of the protein kinase C inhibitors TMB-8 (10 μmol/l), W7, H7, polymyxin-B and sphingosine (all 1 μmol/l) was found to inhibit the steroidogenic response to trypsin. Aldosterone and 18-hydroxycorticosterone were strongly, and corticosterone moderately, affected, while the production of 18-hydroxydeoxycorticosterone was neither stimulated by trypsin nor inhibited by the protein kinase C inhibitors. Addition of neomycin, which prevents substrate interaction with phospholipase C, also inhibited the response to trypsin, while addition of phospholipase C itself stimulated aldosterone, 18-hydroxycorticosterone and corticosterone production with the same tissue sensitivity as trypsin. Addition of phospholipase A2 had no effect. Direct assay of protein kinase C activity showed that trypsin stimulation effected the translocation of Ca2+/phospholipid-activated protein kinase C from the cytosolic to the membrane fraction. When glomerulosa tissue was incubated with [32P]ATP, and cytosolic proteins were subjected to isoelectric focusing on polyacrylimide gels, autoradiography showed that incorporation of 32P into several protein components was increased by trypsin stimulation.

It was concluded that trypsin exerts its stimulatory effects on steroidogenesis by activating protein kinase C; not, however, by generating the Ca2+/phospholipid-independent fragment, but possibly by enhancing the activity of phospholipase C.

Free access

J Liu, X-D Li, A Vaheri, and R Voutilainen

Aberrant DNA methylation may be involved in human adrenocortical tumorigenesis, which is often accompanied by abnormal hormone production. In this study, we aimed to clarify the effects of DNA methylation on steroidogenesis using the human adrenocortical NCI-H295R cell line as a model. Treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (Azad; 10 μM for 7 days) decreased the proliferation rate to approximately 20% and the cell number to 60% of the control, with a simultaneous increase in the expression of the cyclin-dependent kinase inhibitor p57KIP2 gene. In addition, Azad treatment increased cortisol secretion dose and time dependently, whereas dehydroepiandrosterone sulfate secretion was not affected. Azad treatment decreased basal and (Bu)2cAMP-induced expression of low- and high-density lipoprotein receptor, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme, steroid 17α-hydroxylase/17,20-lyase and steroid 21-hydroxylase mRNA, as well as the StAR protein level. In contrast, Azad treatment increased the basal expression of steroid 11β-hydroxylase and 3β-hydroxysteroid dehydrogenase/Δ54-isomerase genes, although it inhibited the (Bu)2cAMP-induced expression of these two genes. The expression of steroidogenic factor-1 (SF-1) and DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome 1) genes (both harboring putative CpG islands in their promoters) and the methylation degree of the HpaII recognition site(s) in the SF-1 gene promoter region were reduced by Azad treatment. The immunostaining pattern of the methyl-CpG-binding protein MeCP2 was also modified by Azad treatment. These results suggest that DNA methylation may be implicated in the regulation of cell proliferation and steroidogenesis in human adrenocortical cells.

Open access

Gillian A Gray, Christopher I White, Raphael F P Castellan, Sara J McSweeney, and Karen E Chapman

Corticosteroids influence the development and function of the heart and its response to injury and pressure overload via actions on glucocorticoid (GR) and mineralocorticoid (MR) receptors. Systemic corticosteroid concentration depends largely on the activity of the hypothalamic–pituitary–adrenal (HPA) axis, but glucocorticoid can also be regenerated from intrinsically inert metabolites by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), selectively increasing glucocorticoid levels within cells and tissues. Extensive studies have revealed the roles for glucocorticoid regeneration by 11β-HSD1 in liver, adipose, brain and other tissues, but until recently, there has been little focus on the heart. This article reviews the evidence for glucocorticoid metabolism by 11β-HSD1 in the heart and for a role of 11β-HSD1 activity in determining the myocardial growth and physiological function. We also consider the potential of 11β-HSD1 as a therapeutic target to enhance repair after myocardial infarction and to prevent the development of cardiac remodelling and heart failure.

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T. Imai, H. Seo, Y. Murata, M. Ohno, Y. Satoh, H. Funahashi, H. Takagi, and N. Matsui

ABSTRACT

The changes in steady-state levels of mRNA for cholesterol side-chain cleavage cytochrome P-450 (P-450scc) and steroid 21-hydroxylase cytochrome P-450 (P-450c21) caused by hypophysectomy and ACTH treatment were determined in rat adrenals. Hypophysectomy caused marked decreases in adrenal weight and total RNA per gland. Administration of ACTH resulted in increases in adrenal weight and total RNA. A significant correlation between the amount of RNA and adrenal weight was observed. Both P-450scc and P-450c21 mRNAs were decreased by hypophysectomy and increased by ACTH treatment. P-450scc mRNA decreased to 20% and P-450c21 mRNA to 76% of control values 1 day after hypophysectomy. ACTH caused a significant increase in P-450scc mRNA after 3 h. However, a significant increase in P-450c21 mRNA was observed 12 h after administration of ACTH. These results are concordant with previous studies in vitro utilizing cultured adrenocortical cells. Moreover, the induction of steady-state levels of P-450scc mRNA was faster than that observed by other investigators in studies in vitro. These results may indicate that integrity of the adrenal gland in vivo is important for the action of ACTH.