Effects of dehydroepiandrosterone on aldosterone release in rat zona glomerulosa cells

The present study was to investigate the effects and action mechanisms of dehydroepiandrosterone (DHEA) on steroidogenesis in rat adrenal zona glomerulosa cells (ZG). ZG cells were incubated with DHEA in the presence or absence of angiotensin II (AngII), a high concentration of potassium, 8-Br-cAMP, forskolin, 25-OH-cholesterol, pregnenolone, progesterone, deoxycorticosterone, corticosterone, A23187, or cyclopiazonic acid (CPA) at 37°C for 1 h. The concentration of aldosterone or pregnenolone in the culture medium was then measured by radioimmunoassay (RIA). The cells were used to determine the cellular cAMP content. The data demonstrated that: (1) DHEA inhibited AngII-, high concentration of KCl-, forskolin-, 8-Br-cAMP-, 25-OH-cholesterol-, pregnenolone-, progesterone-, deoxycorticosterone-, corticosterone-, A23187-, or CPA-stimulated aldosterone release; (2) DHEA increased 25-OH-cholesterol-stimulated pregnenolone release but not when 25-OH-cholesterol was combined with trilostane; (3) DHEA noncompetitively inhibited aldosterone synthase but showed uncompetitive inhibition of P450scc. These results suggest that DHEA acts directly on rat ZG cells to diminish aldosterone secretion by inhibition of a post-cAMP pathway or by acting on intracellular Ca²⁺ mobilization. In addition it affects the function of post-P450scc steroidogenic enzymes. Ling-Ling Chang and Paulus S. Wang contributed equally to this work.     

Effects of prolactin on aldosterone secretion in rat zona glomerulosa cells

Acute effects and action mechanisms of prolactin (PRL) on aldosterone secretion in zona glomerulosa (ZG) cells were investigated in ovariectomized rats. Administration of ovine PRL (oPRL) increased aldosterone secretion in a dose-dependent manner. Incubation of [3H]-pregnenolone combined with oPRL increased the production of [3H]-aldosterone and [3H]-deoxycorticosterone but decreased the accumulation of [3H]-corticosterone. Administration of oPRL produced a marked increase of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in ZG cells. The stimulatory effect of oPRL on aldosterone secretion was attenuated by the administration of angiotensin II (Ang II) and high potassium. The Ca2+ chelator, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 10(-2) M), inhibited the basal release of aldosterone and completely suppressed the stimulatory effects of oPRL on aldosterone secretion. The stimulatory effects of oPRL on aldosterone secretion were attenuated by the administration of nifedipine (L-type Ca2+ channel blocker) and tetrandrine (T-type Ca2+ channel blocker). These data suggest that the increase of aldosterone secretion by oPRL is in part due to (1) the increase of cAMP production, (2) the activation of both L- and T-type Ca2+ channels, and (3) the activation of 21-hydroxylase and aldosterone synthase in rat ZG cells.     

Effect of rat plasma lipoproteins on aldosterone production by rat zona glomerulosa cells in vitro

The possible role of plasma lipoproteins in steroidogenesis by zona glomerulosa cells was studied by examining the effect of rat plasma lipoproteins on aldosterone production by isolated rat zona glomerulosa cells. Neither very low density lipoprotein nor high density lipoprotein caused any increase in either basal or K⁺-stimulated aldosterone production. Low density lipoprotein (LDL) had no effect on basal aldosterone production but had a biphasic effect on K⁺-stimulated aldosterone production, causing a consistent enhancement of steroidogenesis at LDL cholesterol concentrations of 30–60 μg/ml. These data suggest that LDL may play a role in vivo in the supply of cholesterol for steroidogenesis by the rat zona glomerulosa cell.     

Factors affecting the trypsin induced release of aldosterone in rat adrenal zona glomerulosa tissue

The yields of aldosterone obtained during incubation of whole adrenal capsule tissue from the rat (consisting of the connective tissue capsule itself, all of the glomerulosa tissue, and some fasciculata) cannot apparently be accounted for by the gland's capacity for de novo synthesis of this steroid. Recent studies with proteolytic enzymes and inhibitors suggest that in part aldosterone output may result from the activation of proteolytic events which release aldosterone from a sequestered intraglandular pool. These proteolytic events are mimicked by the addition of trypsin to whole tissue incubations in vitro. Experiments were carried out to determine what factors may govern the size of such intraglandular steroid pools. The most remarkable effect was that prior sodium depletion greatly enhanced the yield (2-3-fold) of aldosterone on subsequent incubation of adrenal capsules with trypsin, to an extent far greater than the increase in basal (non trypsin induced) aldosterone output in this tissue. Although betamethasone (20 micrograms/ml in drinking water) and the converting enzyme inhibitor captopril (7.2 mg/day) eliminated trypsin releasable steroid in control animals, they had no effect on the enhanced levels of trypsin releasable steroid seen with sodium depletion. The data suggest that trypsin releasable steroid pools are variable in accordance with the physiological requirements of the animal, particularly in sodium depletion.     

Role of voltage-gated calcium channels in potassium-stimulated aldosterone secretion from rat adrenal zona glomerulosa cells

The mineralocorticoid aldosterone plays an important role in the regulation of plasma electrolyte homeostasis. Exposure of acutely isolated rat adrenal zona glomerulosa cells to elevated K(+) activates voltage-gated calcium channels and initiates a calcium-dependent increase in aldosterone synthesis. We developed a novel 96-well format aldosterone secretion assay to rapidly evaluate the effect of known T- and L-type calcium channel antagonists on K(+)-stimulated aldosterone secretion and better define the role of voltage-gated calcium channels in this process. Reported T-type antagonists, mibefradil and Ni(2+), and selected L-type antagonist dihydropyridines, inhibited K(+)-stimulated aldosterone synthesis. Dihydropyridine-mediated inhibition occurred at concentrations which had no effect on rat alpha1H T-type Ca(2+) currents. In contrast, below 10 microM, the L-type antagonists verapamil and diltiazem showed only minimal inhibitory effects. To examine the selectivity of the calcium channel antagonist-mediated inhibition, we established an aldosterone secretion assay in which 8Br-cAMP stimulates aldosterone secretion independent of extracellular calcium. Mibefradil remained inhibitory in this assay, while the dihydropyridines had only limited effects. Taken together, these data demonstrate a role for the L-type calcium channel in K(+)-stimulated aldosterone secretion. Further, they confirm the need for selective T-type calcium channel antagonists to better address the role of T-type channels in K(+)-stimulated aldosterone secretion.     

In vitro effects of insulin on aldosterone production in rat zona glomerulosa cells.

Though long standing diabetes mellitus is frequently accompanied by hypoaldosteronism, the role of insulin in this setting has never been clearly established. In the present study we have examined the direct effects of insulin on aldosterone production in rat zona glomerulosa cells in vitro. Insulin is shown to directly stimulate aldosterone production in a dose dependent manner, and to attenuate angiotensin II mediated aldosterone production, without affecting angiotensin II receptor binding kinetics. Insulin had no effect on aldosterone production mediated by the other physiological stimuli (K+ and ACTH). These data suggest a possible interaction between insulin and angiotensin II in the regulation of aldosterone secretion.     

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.     

Effect of protein kinase inhibitors on ACTH-stimulated aldosterone production in rat zona glomerulosa cells.

In order to obtain further evidence for the involvement of protein kinases in the short-term ACTH-stimulated aldosterone synthesis in rat zona glomerulosa cells, the effects of three different compounds with protein kinase inhibitory properties were investigated. Staurosporine, H-7 and trifluoperazine inhibited ACTH-stimulated aldosterone release in a dose-dependent manner. While the inhibitory effect of H-7 was reversible upon washing of the cells with inhibitor-free medium, the inhibition was maintained in cells treated with staurosporine or trifluoperazine. In contrast to the stimulated production, basal release of aldosterone even at the highest drug concentrations tested was not completely inhibited. We thus conclude that protein kinases may play a crucial role in short-term ACTH-stimulated aldosterone production in rat glomerulosa cells.     

Effect of the endothelins on aldosterone secretion by rat zona glomerulosa cells in vitro.

Endothelins are thought to be involved in the local regulation of blood flow and tissue function. These experiments were carried out to investigate the possible role of endothelins in the control of aldosterone secretion by the rat adrenal. Suspensions of zona glomerulosa cells were prepared by collagenase digestion of capsular tissue, and incubated in the presence of increasing concentrations of endothelin. Aldosterone was measured by RIA. All three peptides caused a dose-dependent increase in the secretion rate of aldosterone by zona glomerulosa cells. The minimum concentration of peptide required to give a significant response was 10(-14) mol/l for endothelins 2 and 3 and 10(-13) mol/l for endothelin 1. At a concentration of 10(-7) mol/l endothelin 2 elicited a 20-fold increase over basal aldosterone secretion, while both endothelins 1 and 3 elicited a 30-fold increase (P less than 0.001 in all cases). These results show that the endothelins are potent stimulators of aldosterone secretion, and suggest that these peptides may have a role in the control of zona glomerulosa function.     

Isolation of aldosterone synthase cytochrome P-450 from zona glomerulosa mitochondria of rat adrenal cortex

A cytochrome P-450 capable of producing aldosterone from 11-deoxycorticosterone was purified from the zona glomerulosa of rat adrenal cortex. The enzyme was present in the mitochondria of the zona glomerulosa obtained from sodium-depleted and potassium-repleted rats but scarcely detected in those from untreated rats. It was undetectable in the mitochondria of other zones of the adrenal cortex from both the treated and untreated rats. The cytochrome P-450 was distinguishable from cytochrome P-45011 beta purified from the zonae fasciculata-reticularis mitochondria of the same rats. Molecular weights of the former and the latter cytochromes P-450, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 49,500 and 51,500, respectively, and their amino acid sequences up to the 20th residue from the N terminus were different from each other at least in one position. The former catalyzed the multihydroxylation reactions of 11-deoxycorticosterone giving corticosterone, 18-hydroxydeoxycorticosterone, 18-hydroxycorticosterone, and a significant amount of aldosterone as products. On the other hand, the latter catalyzed only 11 beta- and 18-hydroxylation reactions of the same substrate to yield either corticosterone or 18-hydroxydeoxycorticosterone. Thus, at least two forms of cytochrome P-450, which catalyze the 11 beta- and 18-hydroxylations of deoxycorticosterone, exist in rat adrenal cortex, but aldosterone synthesis is catalyzed only by the one present in the zona glomerulosa mitochondria.     

Dopamine inhibition of potassium-stimulated aldosterone biosynthesis in bovine adrenal zona glomerulosa cells

Dopamine inhibits angiotensin II-stimulated aldosterone production by an effect on the late phase of biosynthesis. This study was undertaken to investigate the effect of dopamine on potassium-stimulated aldosterone biosynthesis in adrenal glomerulosa cells in vitro. As potassium concentrations were increased from 0 to 12 mM, aldosterone production increased up to 6 mM potassium, but not beyond this concentration. Dopamine (10(-5)M) inhibited the aldosterone response to potassium. The effect of potassium on pregnenolone accumulation (the early phase of aldosterone biosynthesis) was assessed in cells treated with trilostane which inhibits the conversion of pregnenolone onward to aldosterone. Increasing potassium concentrations up to 12 mM gave increasing pregnenolone accumulation; however dopamine did not influence this effect. The potassium stimulated conversion of corticosterone to aldosterone, an index of activity in the late phase of aldosterone biosynthesis, was assessed using aminoglutethimide to prevent cholesterol side-chain cleavage. Significantly more corticosterone was converted to aldosterone at 6 mM potassium than at 0 or 12 mM; dopamine inhibited the conversion of corticosterone to aldosterone at 6 mM potassium. These data indicate that dopamine inhibits potassium-stimulated aldosterone production by an effect restricted to the late phase of the aldosterone biosynthetic pathway similar to its previously established effect on angiotensin II-stimulated aldosterone biosynthesis.     

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.     

Effects of proteolytic enzymes on steroid release from rat adrenal zona glomerulosa tissue: Evidence for novel steroid-protein complexes

Trypsin (2mg/ml) added to conventional incubations of rat adrenal capsules (largely glomerulosa) reproducibly increases the amount of free extractable aldosterone (aldo) and 18-hydroxycorticosterone (18-OH-B) in incubation media, but has no effect on capsule cell suspensions formed by collagenase incubation. The effect is abolished by the addition of a trypsin inhibitor, but is still seen in the absence of $\text{de novo}$ steroidogenesis. Qualitatively similar results were obtained with capsule homogenates and high speed supernatant fractions, and chromatography of the high speed supernatant protein fraction on Sephadex G-50 gave a number of minor fractions and one major fraction which yielded free aldo on incubation with trypsin. The results indicate the existence of storage forms of aldo and 18-OH-B which are extremely tightly bound to protein. Such steroid-protein complexes appear to be of an entirely novel kind, and are quite distinct from the familiar receptor type complexes. The findings support previously proposed mechanisms for aldo synthesis and secretion.     

Effect of catecholamine on aldosterone release in isolated rat glomerulosa cell suspensions

Effect of adrenergic activity on the adrenal steroidogenesis and the modulation by catecholamines of aldosterone release were studied in isolated rat adrenal cell suspensions. Isoproterenol, norepinephrine and epinephrine, but not dopamine, caused statistically significant increase in aldosterone release. Both prazosin (alpha 1 antagonist) and yohimbine (alpha 2 antagonist) suppressed the norepinephrine-induced aldosterone release in a dose dependent manner, respectively. Both atenolol (beta 1 antagonist) and ICI 118-551 (beta 2 antagonist) also blocked (-)-isoproterenol-induced aldosterone release in a dose dependent manner, respectively. Neither (-)-isoproterenol nor (+/-)-norepinephrine at concentrations of 10(-6) M potentiated aldosterone release stimulated by angiotensin II or ACTH. These results suggest that catecholamines stimulate aldosteroidogenesis, but it appears unlikely that aldosterone release induced by ACTH or angiotensin-II is modulated by adrenergic stimulation.     

Mechanism of action of aldosterone release by prostaglandin E in isolated rat adrenal glomerulosa cells

The effect of prostaglandin E (PGE) on aldosterone release and the mechanism of action of PGE in mediating the release of aldosterone were studied using isolated rat glomerulosa cells. PGE1 stimulated aldosterone release in a dose-dependent fashion at concentrations between 10(-8) and 10(-6) M and caused approximately a two-fold increase over the basal aldosterone level at 10(-6) M. A significant and dose-dependent increase in cAMP production was also produced by PGE1 at concentrations greater than 10(-8) M. Aldosterone release induced by 10(-7) M or 10(-6) M PGE2 was significantly reduced by a competitive receptor blocking PG-antagonist, SC 19220 (10(-7) M), but not affected by (Sar1, Ileu8)-angiotensin-II (A-II), a competitive inhibitor of A-II. PGE-stimulated aldosterone release was almost completely abolished by depleting the extracellular Ca2+ by EGTA, or by verapamil, a Ca2+-channel blocker or W-7, a calmodulin inhibitor. These findings suggest that PGE stimulates aldosterone release through the membrane receptor binding and activation of adenylate cyclase and that Ca2+-calmodulin system plays an essential role in mediating the steroidogenic action of PGE in the adrenal glomerulosa cells. However, the physiological significance of PGE in the regulation of aldosterone secretion remains to be elucidated.     

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.