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.     

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 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.     

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.     

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.     

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.     

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.     

Direct effects of heparin on basal and stimulated aldosterone production in rat adrenal glomerulosa cells

Direct effects of heparin (0.1-10 IU/ml) on basal and stimulated aldosterone production have been studied using intact rat adrenal glomerulosa cells. Heparin at any dose did not affect basal aldosterone production when added to the incubation medium. Heparin at a 0.01 IU/ml dose had no effect on aldosterone production maximally stimulated by angiotensin II (AII, 4.8 X 10(-8) M), ACTH (4.3 X 10(-9) M) or potassium (8.0 mM). However, heparin at 0.1 and 0.3 IU/ml doses selectively blocked aldosterone production maximally stimulated by AII but not by ACTH or potassium, while the compound at 1 and 10 IU/ml doses inhibited aldosterone production maximally stimulated by these three stimuli. In addition, the inhibitory effect of 0.3 IU/ml heparin occurred as early as 30 min after incubation with heparin. These data suggest that heparin at 0.1 and 0.3 IU/ml doses acts directly on adrenal zona glomerulosa to selectively block the stimulatory action of AII, while the compound at 1 and 10 IU/ml doses inhibits all the stimulatory actions of AII, ACTH and potassium.     

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.     

Dopaminergic control of aldosterone: modulation of the response of rat adrenal zona glomerulosa cells to alpha-Msh by pretreatment with bromocriptine or metoclopramide

Bromocriptine treatment in rats (3 mg/kg per day, 7 days) significantly reduced alpha-msh and aldosterone plasma levels 2 hrs after the final treatment in animals on low, normal and high sodium diets. Alpha-MSH dose response curves for corticosterone and 18-hydroxydeoxycorticosterone (18-OH-DOC) in subsequently incubated glomerulosa cells gave stimulation at lower concentrations of alpha-MSH (10(-10) moles per litre) than in cells from untreated animals (10(-9) moles per 1). Curves for aldosterone (ald) and 18-hydroxycorticosterone (18-OH-B) were also affected in cells from animals on a low sodium diet. Fasciculata-reticularis cell responses to ACTH were unaffected. Metoclopramide (4 mg/kg per day, 7 days) elevated plasma alpha-MSH, although ald was unaffected, but inhibited the glomerulosa cell response to alpha-MSH in vitro. Acute dopaminergic responses in plasma ald may be mediated through alpha-MSH in rats, but chronically alpha-MSH may down- regulate glomerulosa cell alpha-MSH receptors. It is unlikely that alpha-MSH mediates the adrenocortical response to sodium depletion.     

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.     

The role of cyclic AMP in aldosterone production by isolated zona glomerulosa cells.

The role of cyclic AMP in the regulation of aldosterone production by adrenocorticotropic hormone (ACTH), angiotensin II (A II), potassium, and serotonin was examined in collagenase-dispersed adrenal glomerulosa cells. The ability of 8-bromo cyclic AMP and choleragen to stimulate maximum aldosterone production indicated that cyclic AMP could act as second messenger for certain of the aldosterone-stimulating factors. The actions of ACTH and choleragen on aldosterone and cyclic AMP production were correlated in dog and rat cells, and a similar relation was seen during stimulation of rat cells by serotonin. In contrast, A II and potassium did not cause changes in cyclic AMP formation while stimulating aldosterone production. Intracellular and receptor-bound cyclic AMP were increased 3-fold by 10(-7) M ACTH but not by A II. Addition of a phosphodiesterase inhibitor increased the magnitude of the cyclic AMP response to ACTH but did not change the lack of stimulation by A II or potassium. In dog cells, the effects of A II and potassium on aldosterone production were partially additive to those of ACTH, choleragen, and 8-bromo cyclic AMP. In contrast, no additivity was observed between A II and potassium, or between combinations of the cyclic AMP-dependent stimuli. These results indicate that the actions of ACTH on aldosterone secretion are mediated by cyclic AMP formation, whereas A II and potassium stimulate aldosterone production through an independent mechanism. The lack of additivity between steroid responses to A II and potassium suggests that these factors could share a common mode of action on steroidogenesis in zona glomerulosa cells.     

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.     

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.     

Effects of protease inhibitors on adenylate cyclase activation and aldosterone production in rat adrenal zona glomerulosa cells

It has been shown that serine proteases are involved in aldosterone and 18-hydroxycorticosterone production by the rat adrenal zona glomerulosa in response to a variety of stimulants. From evidence presented for various tissues, including the rat adrenal cortex, the observation that adenylate cyclase can be activated by proteolytic enzymes and inhibited by protease inhibitors has led to the suggestion that serine proteases may also be involved in the hormonal stimulation of adenylate cyclase. In studies designed to test this hypothesis using protease inhibitors, only high concentrations (greater than 10(-4) M) of TAME (p-tosyl-L-arginine methyl ester) inhibited ACTH stimulated steroid and cAMP production in rat adrenal glomerulosa cells. TPCK (tosyl-L-phenylalanine chloromethylketone) and TLCK (tosyl-L-lysine chloromethylketone) were found to have a similar effect at very high concentrations (10(-2) M) but had no effect at the serine protease inhibitory concentration of 5 X 10(-6) M. Other protease inhibitors tested had no effect on ACTH-stimulated cAMP but the inhibitory effect of high concentrations of protease inhibitors on ACTH-stimulated adenylate cyclase was duplicated by the polyanion dextran sulphate. The results suggest that the inhibitors act through non-specific membrane effects and that proteases are not involved in the activation of zona glomerulosa adenylate cyclase by ACTH. In view of these findings it is concluded that a more rigorous approach should be applied to the use of protease inhibitors in whole cell systems, and that the concept of hormonal activation of adenylate cyclase via proteolytic events, which is based on studies with such inhibitors, should be reconsidered.     

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.     

Effects of ACTH and calcium on cyclic AMP production and steroid output by the zona glomerulosa of the adrenal cortex.

Effects of ACTH and calcium on cyclic AMP production and steroid output by the zona glomerulosa (the capsular fraction) from the rat adrenal cortex have been studied. Although high concentrations of extracellular calcium potentiated the stimulatory action of ACTH on cyclic AMP and aldosterone output, tetracaine or verapamil inhibited aldosterone output but not cyclic AMP production during ACTH-stimulation. Lanthanum reduced both aldosterone and cyclic AMP accumulation induced by ACTH. These results suggest that an extracellular calcium would be essential in stimulating the capsular steroidogenesis without involvement of the cyclic AMP system.     

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.     

Failure of somatostatin and beta-endorphin to affect bovine adrenal cortex cells in vitro

The main intention of this study was to examine the effect of cyclic somatostatin (SRIF) on bovine adrenal cell activity in vitro. beta-Endorphin was applied in another course of experiments. Basal aldosterone secretion when measured was found to be 0.86 +/- 0.33 ng per 10(5) cells and 1.5 h incubation time in the glomerulosa cell fractions. Cortisol production was 1.6 +/- 0.3 ng per 10(5) cells in the fasciculata/reticularis cell incubates respectively. No effect was observed when cyclic somatostatin (SRIF) was added to cell incubates of the bovine adrenal cortex. However, aldosterone secretion was increased 4.5-fold in the presence of 10(-9)M ACTH or 10(-8)M At II. Cortisol production was enhanced similarly after addition of 10(-9)M ACTH.