Interaction of ACTH, beta-endorphin and alpha-melanocyte stimulating hormone in relation to the corticosteroid production of isolated rat adrenocortical zona fasciculata and zona glomerulosa cells.

The combined effects of ACTH, beta-endorphin (beta-EP) and alpha-MSH were studied on the corticosteroidogenesis of isolated rat adrenocortical zona fasciculata and zona glomerulosa cells. beta-EP potentiated the effects of ACTH and alpha-MSH on the zona fasciculata corticosterone production but inhibited those on the zona glomerulosa aldosterone production. beta-EP did not affect the combined action of 4 x 10(-11) M ACTH and 5 x 10(-9) M alpha-MSH on the zona fasciculata or the zona glomerulosa cells, but it inhibited the stimulatory action of the combination of 1.6 x 10(-10) M ACTH and 10(-9) M alpha-MSH on the zona glomerulosa aldosterone production. An interaction of ACTH, beta-EP and alpha-MSH in relation to the zona fasciculata and zona glomerulosa corticosteroid production was found.     

Role of chain termini in selective steroidogenic effect of ACTH/MSH(4-10) on isolated adrenocortical cells

To investigate the role of charged chain ends in the corticosteroidogenic effect of ACTH/MSH(4-10), acetyl and amide derivatives of ACTH/MSH(4-10) were synthesized and tested in isolated zona glomerulosa and zona fasciculata cells. ACTH/MSH(4-10)-NH2, Ac-ACTH/MSH(4-10) and Ac-ACTH/MSH(4-10)-NH2 (10 microM to 1 mM) stimulated the aldosterone production of zona glomerulosa cells, whereas these peptides did not stimulate the corticosterone production of zona fasciculata cells, even at 1 mM concentration. As ACTH/MSH(4-10) has been shown to have a steroidogenic effect on both types of adrenocortical cells, both charged chain termini seem to be essential for triggering of the corticosterone production of zona fasciculata cells, but for aldosterone production their presence appears not to be important.     

Effect of pituitary intermediate lobe extract on steroid production by the isolated zona glomerulosa and fasciculata cells

The effect of intermediate lobe extract (ILE) on aldosterone and corticosterone production of the zona glomerulosa cells and on corticosterone production of the zona fasciculata cells was investigated. The slope of the dose-response curve of ILE dilution was steeper than that of alpha h 1-39 ACTH measured on zona glomerulosa steroid production. The ED50 of both ILE and ACTH was lower when measured on zona glomerulosa than on zona fasciculata steroid production. It is supposed that a hormone (or some other substance) in ILE alters the sensitivity to ACTH of the zona glomerulosa cells.     

Structure-activity studies with ACTH/alpha-MSH fragments on corticosteroid secretion of isolated zona glomerulosa and fasciculata cells

The steroidogenic action of ACTH/alpha-MSH fragments was studied on isolated zona glomerulosa and zona fasciculata cells dispersed by collagenase. ACTH-(4-7), ACTH-(6-10), ACTH-(4-10) and ACTH-(11-13) stimulated corticosterone production of the zona fasciculata and aldosterone production of the zona glomerulosa cells. ACTH-(7-10) was ineffective. ACTH-(4-7) appeared to be the most potent peptide of the tested fragments. None of the fragments affected the steroidogenic action of ACTH-(1-39). It is suggested that similar to the melanotropic effect of alpha-MSH two 'message' sequences for adrenocortical stimulation exist in the alpha-MSH part of the ACTH molecule.     

Effect of alpha-MSH on the corticosteroid production of isolated zona glomerulosa and zona fasciculata cells

α-MSH (10^?9 ? 6×10^?7M) potentiates the effect of ACTH (10^?11 ? 5×10^?9M) on adrenocortical steroidogenesis decreassng ED₅₀ of ACTH from 220 to 183 pg/ml on zona fasciculata corticosterone-, and from 739 to 437 pg/ml on zona glomerulosa aldosterone production. α-MSH alone increases aldosterone production of zona glomerulosa cells in doses (10^?9 ? 6×10^?7M) that do not stimulate zona fasciculata corticosterone production. The response of zona glomerulosa aldosterone production to α-MSH can be characterized by a bi-phase dose-response curve.     

Calf adrenocortical fasciculata cells secrete aldosterone when placed in primary culture

Aldosterone production occurs in the outer area of the adrenal cortex, the zona glomerulosa. The glucocortocoids cortisol and corticosterone, depending upon the species, are synthesized in the inner cortex, the zona fasciculata. Calf zona glomerulosa cells rapidly lose the ability to synthesize aldosterone when placed in primary culture unless they are incubated in the presence of the antioxidants butylated hydroxyanisol and selenous acid, the radioprotectant DMSO, and the cytochrome P-450 inhibitor metyrapone. In the presence of these additives, calf zona fasciculata cells in primary culture synthesize aldosterone at rates which can approach those from cells isolated from the zona glomerulosa. Calf zona glomerulosa and fasciculata cells both responded well to ACTH and angiotensin II, but the zona fasciculata cells respond very poorly compared to glomerulosa cells to increased potassium in the media. Rat zona fasciculata cells in primary culture under similar conditions did not synthesize aldesterone, suggesting that the regulation of the expression of the enzymes responsible for the biosynthesis of aldosterone in the two species is different. Two distinct cytochrome P-450 cDNAs which hydroxylate deoxycorticosterone at the 11β position have been described in the rat, human and mouse. Both cytochrome P-450 cDNAs have been cloned and expressed in non-steroidogenic cells, but only one is expressed in the zona glomerulosa and only this glomerulosa cytochrome P450 can further hydroxylate deoxycorticosterone to generate aldosterone. Two bovine adrenal cDNAs have been described with 11β-hydroxylase activity and their expression products in transiently transfected COS cells can convert deoxycorticosterone into aldosterone. Both enzymes are expressed in all zones of the adrenal cortex. Zonal regulation of aldosterone synthesis in the bovine adrenal gland may be due to an 11β-hydroxylase with aldosterone synthesizing capacity which has not yet been isolated. Alternatively, a single enzyme might be responsible for the several hydroxylations in the pathway between deoxycorticosterone and aldosterone and zonal synthesis might be controlled by unknown factors regulating the expression of C-18 hydroxylation. The incubation of zona fasciculata with antioxidants and metyrapone results in atypical expression of this activity by an unclear mechanism.     

The inhibitor of phospholipase-A2, AACOCF3, stimulates steroid secretion by dispersed human and rat adrenocortical cells

AACOF3 is a trifluomethylketone analog of arachidonic acid, which inhibits phospholipase-A2 (PLA2). AACOCF3 was found to concentration-dependently increase basal aldosterone and corticosterone secretion by dispersed rat zona glomerulosa and zona fasciculata/reticularis cells, respectively, as well as aldosterone and cortisol production by dispersed human adrenocortical cells. Maximal effective concentration was 10(-5) M, and elicited about 2.5-3.0-fold rises in steroid output. 10(-5) M AACOCF3 also enhanced submaximally (10(-15)/10(-12) M), but not maximally (10(-9) M) ACTH-stimulated hormonal secretion. Quantitative HPLC showed that 10(-5) M AACOCF3 evokes similar increases (from 2.0- to 3.0-fold) in the basal release of the entire spectrum of adrenocortical steroids (i.e. both intermediate and definitive products of steroid synthesis), thereby suggesting that AACOCF3 acts on the early steps of steroid synthesis. Accordingly, when pregnenolone metabolism is prevented by cyanoketone, 10(-5) M AACOCF3 increased by about 8-10-fold the production of this steroid. In conclusion, we have demonstrated a side-effect of AACOCF3, which may become relevant in studies where this chemical is used to inhibit PLA2 in tissues able to convert cholesterol to pregnenolone.     

Effect of beta-endorphin on the steroid production of isolated zona glomerulosa and zona fasciculata cells

Small doses of β-endorphin (10^?11?10^?5M) decrease corticosterone production of zona fasciculata cells but fail to influence steroid production of zona glomerulosa cells. 10^?4M β-endorphin increases corticosterone production of both zones. The stimulating effect of ACTH on zona fasciculata corticosterone- and zona glomerulosa aldosterone production was decreased by β-endorphin (10^?9?10^?7M). Conclusion: β-endorphin might modulate both basal and ACTH stimulated corticosterone secretion.     

Effects of atrial natriuretic peptide AP II on the morphofunctional state of the adrenal cortex of white rats]

The effect of atrial natriuretic peptides synthetic analog AP II on adrenal zona glomerulosa and zona fasciculata physiological regeneration have been studied on male rats. The 3H-thymidine incorporation into DNA in adrenal cortical cells was evaluated in 4 and 24 h after intraperitoneal injection of 10 or 100 mcg/kg AP II. Besides, we have investigated the influence of AP II on adrenal cortical cells karyometric parameter in 4 and 24 h and aldosterone plasma concentration in 1 h after injection. 10 mcg/kg AP II increased the fraction of labelled nuclei in zona glomerulosa and decreased the aldosterone plasma level. No significant changes were seen in zona fasciculata cells proliferation. 100 mcg/kg AP II inhibited the DNA synthesis process in adrenal zona fasciculata, but had no significant influence on zona glomerulosa physiological regeneration and aldosterone plasma concentration. No nuclear morphometric parameter changes were observed in adrenal glomerulosa and fasciculata cells of AP II--treated animals.     

Effects of ACTH-(11-24) on the corticosteroid production of isolated adrenocortical cells

The steroidogenic action of ACTH-(11-24) was studied on isolated zona glomerulosa and zona fasciculata cells dispersed by collagenase. ACTH-(11-24) stimulated the corticosterone production of zona fasciculata cells and the aldosterone production of zona glomerulosa cells; in addition, it potentiated the effects of ACTH-(1-39) on both cell systems. It is suggested that the ACTH molecule contains more active sites for steroidogenesis than usually acknowledged, as has been found for lipolysis and behavior.     

Atrial natriuretic factor inhibits the early pathway of steroid biosynthesis in bovine adrenal cortex

We have previously determined that atrial natriuretic factor (ANF) is a potent inhibitor of steroid secretion in cultured bovine zona glomerulosa and fasciculata cells. The present report describes a comparison of the effect produced by ANF on aldosterone, deoxycorticosterone and progesterone secretions by zona glomerulosa cells and on cortisol, corticosterone and progesterone secretions by zona fasciculata cells. The equipotent inhibitory action of ANF on the stimulated secretion of these steroids in both cell types indicates a common site of action prior to progesterone synthesis at which ANF inhibits the steroidogenic pathway.     

Glucocorticoid suppression enhances the 18-hydroxycorticosterone and aldosterone response to metoclopramide in man

18-Hydroxycorticosterone (18-OHB) is a precursor of aldosterone and is the only corticosteroid, other than aldosterone, that is synthesized predominantly in the zona glomerulosa. Administration of the dopamine antagonist, metoclopramide results in parallel rises in plasma 18-OHB and aldosterone levels without affecting the plasma levels of other aldosterone precursors. However, 18-OHB is a product of the zona fasciculata as well as the glomerulosa. Thus, it is possible that metoclopramide may stimulate zona fasciculata secretion of 18-OHB. In order to more selectively examine dopaminergic regulation of zona glomerulosa secretion of 18-OHB we have examined the effect of glucocorticoid suppression of the fasciculata on the 18-OHB and aldosterone responses to metoclopramide, 10 mg iv in 6 normal volunteers. Dexamethasone, 2 mg every 6 hours for 5 days, suppressed basal levels of cortisol, corticosterone, 18-OHB and aldosterone. Dexamethasone treatment had no effect on basal levels of PRA or PRA responses to metoclopramide. The 18-OHB and aldosterone responses to metoclopramide were enhanced (p less than .05) by dexamethasone suppression. The results suggest that dopaminergic mechanisms selectively suppress glomerulosa production of 18-OHB. Endogenous ACTH may inhibit zona glomerulosa production of 18-OHB and aldosterone in response to the dopamine antagonist, metoclopramide.     

Ultrastructural Characteristics of Adult Rat Adrenocortical Cells Maintained in vitro With and Without ACTH

Bhattacharyya, T. K., Butler, D. G., Price, C. S. 1980. Ultrastructural characteristics of adult rat adrenocortical cells maintained in vitro with and without ACTH. (Ramsay Wright Zoological Laboratories, Department of Zoology, University of Toronto, and Division of Endocrinology, Toronto Western Hospital, and Department of Medicine, University of Toronto, Canada.) — Acta zool. (Stockh.) 61(1): 9–21. The ultrastructural morphology of adrenocortical cells of adult rats maintained in culture for four days with and without corticotropin (ACTH) was studied in comparison to freshly dissociated cells. Identification of cells belonging to zona glomerulosa, zona fasciculata, and zona reticularis was made on the characteristics of the mitochondria and smooth endoplasmic reticulum (SER), and was confirmed by comparison with cellular layers from intact adrenals. Without ACTH, glomerulosa and fasciculata cells showed disappearance of SER, atrophied Golgi apparatus (GA), and a striking proliferation of granular reticulum. Fascicular cells had a loss in mitochondrial matrix density and the mitochondrial cristae showed a tendency to convert to lamellar glomerulosa-type cristae. Zona reticularis cells were not strikingly altered. Maintenance with ACTH led to increased cell size and islet formation of cortical cells. Glomerulosa cells had normal appearance and fasciculata cells manifested a pronounced development of GA and SER, and a normal configuration of mitochondria. The effects of ACTH on glomerulosa cells suggest a trophic influence of ACTH on these cells in vitro. The alterations observed in fascicular cell mitochondria and SER can be explained in terms of known concepts of steroid biosynthesis and basically agree with the patterns of steroid synthesis observed in these cells previously (Price et al. 1975).     

Conditionally immortalized adrenocortical cell lines at undifferentiated states exhibit inducible expression of glucocorticoid-synthesizing genes.

To facilitate studies on differentiation of adrenocortical cells and regulation of steroidogenic genes, we established cell lines from adrenals of adult transgenic mice harboring a temperature-sensitive large T-antigen gene of simian virus 40. Adrenal glands of the mice exhibited normal cortical zonation including a functionally undifferentiated cell-layer between the aldosterone-synthesizing zona glomerulosa cells and the corticosterone-synthesizing zona fasciculata cells. At a permissive temperature (33 degrees C), established cell lines AcA201, AcE60 and AcA101 expressed steroidogenic genes encoding steroidogenic factor-1, cholesterol side-chain cleavage P450scc, and steroidogenic acute regulatory protein, which are expressed throughout adrenal cortices and gonads. Genes encoding 3 beta-hydroxysteroid dehydrogenase and steroid 21-hydroxylase P450c21, which catalyze the intermediate steps for syntheses of both aldosterone and corticosterone, were inducible in the three cell lines in temperature- and/or dibutyryl cAMP-dependent manners. Notably, these cell lines displayed distinct expression patterns of the steroid 11 beta-hydroxylase P45011 beta gene responsible for the zone-specific synthesis of corticosterone. AcA201 cells expressed the P45011 beta gene at 33 degrees C, showing the property of the zona fasciculata cells, while AcE60 cells expressed it upon a shift to a nonpermissive temperature (39 degrees C). On the other hand, AcA101 expressed the P45011 beta gene at 39 degrees C synergistically with exposure to dibutyryl cAMP. None of these clones express the zona glomerulosa-specific aldosterone synthase P450aldo gene under the conditions we tested. These results show that AcE60 and AcA101 cells display a pattern of the steroidogenic gene expression similar to that of the undifferentiated cell-layer and are capable of differentiating into the zona fasciculata-like cells in vitro.     

18-Ethynyl-deoxycorticosterone inhibition of steroid production is different in freshly isolated compared to cultured calf zona glomerulosa cells

The inhibiting effects of 18-ethynyl-deoxycorticosterone (18-E-DOC) as a mechanism-based inhibitor on the late-steps of the aldosterone biosynthetic pathway were examined in calf adrenal zona glomerulosa cells in primary culture and in freshly isolated calf zona glomerulosa cells. 18-E-DOC inhibited the stimulated secretion of aldosterone and 18-hydroxycorticosterone in a similar dose-response and time fashion. No significant differences were found between the inhibition in cultured and freshly isolated cells (K_i of 0.25 vs 0.26 μM) Corticosterone secretion stimulated by ACTH or angiotensin II was also cultured in freshly isolated zona glomerulosa and fasciculata cells, but was not inhibited in cultured calf adrenal cells. Cortisol secretion stimulated by ACTH was not inhibited by 18-E-DOC in cultured zona fasciculata adrenal cells, but was inhibited in freshly isolated zona fasciculata cells with a K_i of 48 μM. The secretion of 18-hydroxyDOC or 19-hydroxyDOC stimulated by ACTH was not inhibited by 18-E-DOC. The bovine adrenal has been reported to have cytochrome P-450 11β-hydroxylases that can perform the various hydroxylations required for the synthesis of cortisol and aldosterone in the different areas of the adrenal. In other species a distinct 11β-hydroxylase which participates in the biosynthesis of aldosterone and is located in the zona glomerulosa has been described. These studies with the mechanism-based inhibitor, 18-E-DOC, suggest that the bovine adrenal functions in a manner very similar to that of other species and raises the possibility that a distinct 11β-hydroxylase with aldosterone synthase activity might be present, but has not been cloned as yet.     

An ultrastructural study of the adrenal cortex in Cushing's syndrome without and with hyperaldosteronism]

This paper reports ultrastructural comparision of the adrenal gland zones of a rare case of a combination from Cushing's syndrome and hyperaldosteronism with a pure Cushing's syndrom and with a normal human adrenal gland. Whilst between the zona glomerulosa of the normal adrenal gland and of the Cushing's syndrom there are no differences, the morphological state of the glomerulosa cells of the Cushing's syndrom in combination with hyperaldosteronism is interpreted as an inhibition of the activity of these cells. In the cells of the hyperplastic zona fasciculata of Cushing's syndrom in combination with hyperaldosteronism, there are no electronmicroscopical characteristics, who are morphological equivalent of the production of aldosterone. The abundant quantity of steroid producing organells in these cells point to a high function. The cells of the adenoma of Cushing's syndrom contain abundant vacuols of lipids, in which presumable are stored steroid hormones.     

Calcium stimulates steroidogenesis in permeabilized bovine adrenal cortical cells

The effect of Ca2+ on steroid production was examined in electropermeabilized bovine adrenal zona glomerulosa and fasciculata cells. The cells were superfused with a medium mimicking cytosolic ionic content but deprived of Ca2+. The permeabilized glomerulosa cells produced aldosterone at a low basal rate. Upon addition of NADP+ to the medium, a transient and concentration-dependent (EC50 = 6 microM) peak of aldosterone production occurred. When the superfusion medium was supplemented with buffered Ca2+ at submicromolar concentrations, a concentration-dependent and sustained increase of aldosterone output was observed. The maximal response (2-3 times the basal secretion rate) was achieved with 1-2 microM ambient free Ca2+, and the EC50 for Ca2+ was 0.5 microM. The continuous presence of NADP+ was found to be necessary for a Ca2+ effect. The Ca2+-induced aldosterone response was entirely blocked by ruthenium red (1 microM), an inhibitor of mitochondrial Ca2+ uptake, and by W-7 (5 microM), a calmodulin inhibitor. Qualitatively and quantitatively similar results were obtained for corticosterone production in adrenal fasciculata cells. These results show that permeabilized adrenal cortical cells retain the ability to produce steroids. Moreover, Ca2+ influx into the mitochondria and Ca2+/calmodulin-dependent reactions appear to be critical steps in the activation of steroidogenesis. These studies provide a further direct link between cytosolic free calcium concentration and biological responses induced by steroidogenic, calcium-mobilizing stimulators in the adrenal cortex.     

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.     

Regulation of adrenocortical steroidogenesis by benzodiazepines

Benzodiazepines affect steroidogenesis in at least four ways depending on concentration and adrenocortical cell type. Firstly, at micromolar concentrations, they inhibit steroidogenic enzymes. Competition for microsomal 17- and 21-hydroxylase activity explains the inhibition of ACTH-stimulated aldosterone and cortisol synthesis by diazepam and midazolam. At slightly higher concentrations, we have evidence that 11β-hydroxylase activity is also inhibited. Secondly, at sub-micromolar concentrations, calcium influx is inhibited. T-type and L-type calcium channels appear to be blocked, this impairs signal response coupling and, in particular, decreases angiotensin-and K⁺-stimulated aldosterone synthesis in zona glomerulosa cells. Thirdly, the mitochondrion of steroidogenic tissues is a sensitive site for the stimulatory effects of benzodiazepines. Aldosterone synthesis from added HDL-cholesterol by cultured bovine zona glomerulosa cells is stimulated by diazepam, RO5-4864 and PK11195. The fourth site of benzodiazepine's effect on steroidogenesis is particular to zona glomerulosa cells. In addition to cholesterol side chain cleavage, the final part of the aldosterone biosynthetic pathway, the conversion from deoxycorticosterone is controlled. Although high micromolar concentrations of diazepam appear to be inhibitory, lower nanomolar concentrations stimulate the synthesis of aldosterone from added deoxycorticosterone. In vivo, a fifth site of benzodiazepine activity may influence plasma steroid concentrations. Competition between steroids and benzodiazepines for hepatic clearance enzymes may affect half lives of both drugs and hormones.