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.     

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 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.     

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.     

Angiotensin II-induced inositol phosphate production in isolated rat zona glomerulosa and fasciculata/reticularis cells

Angiotensin II (2.5 to 250nM) induced, within 60 sec, a significant increase in [3H]inositol-labeled inositol phosphate, inositol bisphosphate, and inositol trisphosphate in rat zona glomerulosa cells. Neither ACTH (3nM) nor K+ (8.4mM) had any effect, although aldosterone and corticosterone were significantly stimulated by all three agonists (after 30 min incubation). A similar significant dose-dependent increase in the inositol phosphates was observed with angiotensin II in zona fasciculata/reticularis cells after 30 min, but without any effect on corticosterone. In contrast ACTH significantly increased corticosterone with only a small although highly significant increase in inositol trisphosphate and inositol bisphosphate at 0.03nM ACTH. However at the higher dose (3.0nM) only inositol bisphosphate was significantly increased. These results indicate the presence on both zona glomerulosa and zona fasciculata/reticularis cells of AII receptors, which were linked to the formation of the secondary messenger, but only in the zona glomerulosa cells are associated with steroidogenesis.     

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.     

Guinea pig adrenocortical cells: in vitro characterization of separated zonal cell types

This paper reports a quick, relatively simple and reproducible technique for obtaining populations of zona fasciculata and zona glomerulosa cells up to 80-90% pure, which can be maintained in vitro for study of adrenocortical cell function. Isolated guinea pig adrenocortical cells were separated on a 1-28% bovine serum albumin/Ca++, Mg++-free buffer gradient (wt/vol at 4% increments) using equilibrium density centrifugation (570 g, 30 min). Over 60% of the 8 x 10(5) viable cells/adrenal obtained in the total isolate were recovered after separation. 80% of the zona glomerulosa cells were found in the lower three bands of the gradient. 78% of the zona fasciculata cells were found in the top three bands. Of the cells in the first two bands, 78-91% were zona fasciculata cells, whereas of the cells in the bottom two bands 92-95% were zona glomerulosa cells. The cells retained the morphological characteristics of cells in situ and could be maintained in vitro for periods up to 11 d. They produced a wide variety of steroids, cortisol, corticosterone, aldosterone, 11-beta- hydroxyandrostenedione, deoxycortisol, deoxycorticosterone, cortisone, 18-hydroxycorticosterone, and a product tentatively identified as dehydroepiandrosterone, and they responded to ACTH in a dose-responsive manner with enhanced levels of steroid output. Zona glomerulosa- enriched populations differed from zona fasciculata-enriched populations in their abundant production of aldosterone and in the pattern of steroid production. None of the cultures responded to angiotensin II (100 pg/ml) with increased steroid production.     

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.     

Leukotrienes as common intermediates in the cyclic AMP dependent and independent pathways in adrenal steroidogenesis

Aldosterone secretion from adrenal glomerulosa cells can be stimulated by angiotensin II (AII), extracellular potassium and adrenocorticotropin (ACTH). Since the mitochondria can recognize factors generated by AII (cyclic-AMP-independent) and ACTH (cyclic AMP dependent), it is reasonable to postulate the existence of a common intermediate in spite of a different signal transduction mechanism. We have evaluated this hypothesis by stimulation of mitochondria from glomerulosa gland with fractions isolated from glomerulosa gland stimulated with AII or from fasciculata gland stimulated with ACTH; the same fractions were tested using mitochondria from fasciculata cells. Postmitochondrial fractions (PMTS) obtained after incubation of adrenal zona glomerulosa with or without AII (10(-7) M) or ACTH (10(-10) M), were able to increase net progesterone synthesis 5-fold in mitochondria isolated from non-stimulated rat zona glomerulosa. In addition, AII in zona glomerulosa produced in vitro steroidogenic fractions that were able to stimulate mitochondria from zona fasciculata cells. Inhibitors of arachidonic acid release and metabolism blocked corticosterone production in fasciculata cells stimulated with ACTH. This concept is supported by the experiment in which bromophenacylbromide and nordihydroguaiaretic acid also blocked the formation of an activated PMTS. In fact, non-activated PMTS, in the presence of exogenous arachidonic acid AA, behaved as an activated PMTS from ACTH stimulated cells. We suggest that the mechanisms of action of ACTH and AII involve an increase in the release of AA and an activation of the enzyme system which converts AA in leukotriene products.     

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.     

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.     

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.     

Aldosterone and corticosterone stimulation by ACTH in isolated rat adrenal glomerulosa cells: interaction with vasopressin

An interaction between ACTH and vasopressin on steroidogenesis was observed in isolated rat adrenal zona glomerulosa cell preparations. 1. The presence of 10(-11) M vasopressin further increased by 52% the output of aldosterone produced by 10(-12) M ACTH on those cells. 2. At a pharmacological concentration of ACTH (10(-7) M), the aldosterone output was increased 5 fold while the addition of 10(-12) M or 10(-8) M vasopressin decreased it by 17% and 48% respectively. 3. Vasopressin also produced a dose-dependent inhibition of the stimulatory effect of ACTH on the output of corticosterone. 4. We have thus shown for the first time, that vasopressin acts directly on adrenal zona glomerulosa cell preparations to modify the aldosterone output by modulating the action of ACTH. It is postulated that, in addition to other known aldosterone regulating factors, ACTH and vasopressin might synergistically act to regulate the secretion of aldosterone in vivo.     

Rat glomerulosa cells in primary culture and E. coli lipopolysaccharide action

During endotoxic shock there is a dysfunction of the adrenal gland; both corticosterone and aldosterone secretion are altered. The aim of the present study is to use glomerulosa cells in primary culture as a target of lipopolysaccharide (LPS) action. Glomerulosa cells cultured in basal conditions are able to proliferate; bFGF and ACTH have antagonic effects, bFGF increases proliferation whereas ACTH is antimitogenic. LPS has a biphasic effect, in the short term it is antimitogenic and in the long term increases the proliferation rate. LPS inhibits ACTH-induced corticosterone secretion in a dose-dependent manner in glomerulosa cells in culture similar to that in fasciculata cells, but it does not exert an important direct effect on aldosterone secretion. These results show that LPS exerts different effects in ACTH and ANG II signal transduction pathways and in the two enzymes which catalyze the late step in the steroidogenesis, 11beta-hydroxylase and aldosterone synthase, which could be in agreement with the existence of both enzymes, regulated independently, in rat zona glomerulosa cells.     

Effect of a cholesterol synthesis inhibitor (BM 15.766) in the presence and absence of HDL on corticosteroidogenesis of isolated zona glomerulosa and fasciculata cells

The effect of the cholesterol synthesis inhibitor BM 15.766, 4-[2-[1-(4-chlorocinamyl)piperazin-4-yl]ethyl]-benzoic acid on the corticosteroid production was studied in order to reveal the importance of endogenous cholesterol synthesis in the function of zona glomerulosa and zona fasciculata cells of rats. Attempts were made to compensate the effect of BM 15.766 through the application of high-density lipoproteins (HDL). Electron microscopy was used to trace the binding and intracellular accumulation of colloidal gold-labelled HDL (HDL-Au, a cholesterol carrier), in the presence of the cholesterol biosynthesis inhibitor. The stimulation of both types of cells with ACTH was less effective in the presence of 2 x 10(-5) M BM 15.766. The inhibitory effect of BM 15.766 was most marked on the aldosterone production of the zona glomerulosa cells, and could not be reversed by addition of a small amount of HDL-Au. Corticosterone-aldosterone conversion was inhibited by 2 x 10(-5) M BM 15.766. ACTH-stimulated, short-term HDL uptake and internalization was not affected by the cholesterol synthesis inhibitor. The results suggest that certain metabolites of de novo cholesterol biosynthesis may participate in the control of aldosterone production.     

Vasopressin: a potent growth factor in adrenal glomerulosa cells in culture

Previous studies have shown that vasopressin stimulates the mitotic activity in adrenal zona glomerulosa cells in intact as well as in hypophysectomized rats. (Payet and Isler, Cell and Tissue Res. 172, 1976; Payet and Lehoux, J. steroid Biochem. 12, 1980). We now report that this effect is direct and specific, since vasopressin stimulates the mitotic activity of rat adrenal zona glomerulosa cells in primary cultures. These cells were prepared by dissociation with collagenase in the culture medium MEM-d-Valine. Isolated cells were placed in 3.5 diameter petri dishes in MEM-d-valine medium containing 15% fetal calf serum and antibiotics for two days and 5% fetal calf serum for subsequent cultures. The medium was changed at 24 hr intervals. The hormones were added 3 days after the culture was started. The mitogenic effect of vasopressin was found to be dependent both on time and hormone concentrations. Vasopressin (10(-11) M) stimulated thymidine incorporation 4.8 +/- 0.6-fold after 2 days of treatment and 5.3 +/- 1.6-fold after 8 days. When ACTH (10(-11) M) was added together with vasopressin (10(-11) M) the mitogenic effect was enhanced at 6.5 +/- 1.9-fold after 2 days and 12.9 +/- 6.9-fold after 8 days of treatment. The aldosterone and corticosterone outputs were also stimulated by the combined presence of vasopressin and ACTH in the incubation medium; a maximal effect was observed between 6 and 8 days of treatment. Vasopressin (10(-11) M) + ACTH (10(-11) M) stimulated the aldosterone output 7-fold and that of corticosterone by 18-fold. When added alone, vasopressin, as well as ACTH alone had only a small effect on the aldosterone output. However, ACTH alone stimulated the corticosterone output 10-fold. In conclusion, vasopressin is an important and specific growth factor of the adrenal zona glomerulosa cells. In addition, together with ACTH vasopressin stimulates the aldosterone and corticosterone output both in vivo and in vitro in primary cell cultures.     

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.     

Effect of ACTH on biosynthesis of aldosterone and corticosterone by monolayer cultures of rat adrenal zona glomerulosa cells

A method is described for preparing monolayer cultures of zona glomerulosa cells isolated from the rat adrenal cortex. Aldosterone and corticosterone were secreted by the cultures when maintained with medium containing 11 mM K⁺. ACTH, while stimulating aldosterone biosynthesis at first, did not maintain its long-term secretion, yet caused corticosterone production to rise to a steadily maintained level. The significance of this effect is discussed.