Dissociation of aldosterone and prostaglandin biosynthesis in the rat adrenal glomerulosa

The relationship between aldosterone production and prostaglandin E₂ synthesis was evaluated using the responses of isolated rat adrenal glomerulosa cells to angiotensin II, ACTH and potassium. Simultaneous PGE₂ and aldosterone measurements were made during timed incubations with these stimuli, and in incubations with arachidonic acid, meclofenamate, indomethacin, and aminoglutethamide. PGE₂ and aldosterone production were assessed by radioimmunoassay. We were not able to demonstrate stimulation of PGE₂ by angiotensin II, ACTH, or potassium despite significant increments in aldosterone production with these stimuli. Arachidonic acid enhanced PGE₂ synthesis, but had no effect on aldosterone release. Indomethacin and meclofenamate inhibited aldosterone secretion. Aminoglutethimide depressed aldosterone production, but had little effect on PGE₂ levels in the media.These studies demonstrate that dienoic prostaglandins play no direct role in aldosterone production stimulated by angiotensin II, ACTH, or potassium in rat adrenal glomerulosa cells. Since inhibitors of cyclo-oxygenase decreased aldosterone synthesis, it is possible that fatty acids other than arachidonic acid may be cyclo-oxygenated to products which regulate aldosterone production.     

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.     

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.     

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.     

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.     

Dopamine in rat adrenal glomerulosa

There is increasing evidence that dopamine (DA) inhibits aldosterone production, but the source of DA for this dopaminergic influence is not known. In the present study we examined the adrenal's zona glomerulosa for the presence of DA. Rats maintained on an intake of regular food were killed by decapitation and the adrenal capsule (containing zona glomerulosa) and the remainder of the gland (containing both cortex and medulla) were examined for their content of DA and also for norepinephrine (NE) and epinephrine (E). DA was found in adrenal glomerulosa in substantial quantity, 1.92 +/- 0.17 (SEM) ng/mg wet weight, representing an approximate concentration of DA of 1-100 microM. DA in adrenal capsule represented 12.2% of the total adrenal content of DA. NE and E were also present in glomerulosa, 3.46 +/- 0.32 and 18.7 +/- 2.1 ng/mg respectively, but, unlike DA, about 98% of the total adrenal content of NE and E was contained in adrenal medulla. The NE/E ratio in capsule and medulla were similar, although slightly higher in adrenal medulla, suggesting that the medulla is the source of the NE and E found in glomerulosa. On the other hand, the DA/E ratio was several-fold higher in glomerulosa than medulla--suggesting that glomerulosa DA was derived at least partially from a source other than adrenal medulla. We also found that short-term culturing of the adrenal reduced DA levels to 1/3 that observed in fresh tissue. This could explain in part why cultured glomerulosa has been shown to be more responsive to administered stimuli. In summary, the findings indicate a significant concentration of DA in adrenal glomerulosa, and suggest that the effects of DA on aldosterone production are mediated locally within the adrenal.     

Diazepam inhibits potassium-induced aldosterone secretion in adrenal glomerulosa cell

In an attempt to elucidate a possible role of peripheral benzodiazepine receptor in adrenal glomerulosa cell, effect of diazepam on potassium-induced aldosterone secretion was studied using isolated bovine adrenal glomerulosa cell. Diazepam inhibited aldosterone secretion stimulated by 8mM potassium in a dose dependent manner. The ID50 was approximately 14 nM. Although diazepam inhibited potassium action effectively, forskolin-induced aldosterone secretion was not affected by diazepam. These results indicate that peripheral benzodiazepine receptor may have an active role in regulating aldosterone secretion. The voltage dependent calcium channel may be a possible site of benzodiazepine action in this tissue.     

Control of aldosterone secretion in zona glomerulosa cell suspensions and in the perfused adrenal gland of the rat

The secretion of aldosterone and its responses to stimulation have been studied in rat adrenal zona glomerulosa tissue incubated as intact capsules or as collagenase-dispersed cell suspensions, and in intact perfused rat adrenal glands. Several differences are apparent in the functions of the various preparations. Aldosterone secretion rates are similar in incubated intact capsules and in the perfused gland. Relative to corticosterone, lower yields of aldosterone are obtained in dispersed glomerulosa cell in vitro. This may be related to the loss in the dispersed cells of a pool of tissue steroid (aldosterone or a precursor) which is revealed only in intact tissue incubations by trypsin stimulation of aldosterone secretion. Trypsin-released aldosterone is increased by prior dietary sodium restriction. In addition, differences occur in the responses of dispersed cells and perfused glands to stimulation. Perfused glands from animals on a normal diet are less sensitive to stimulation by ACTH or alpha-MSH, but more sensitive than dispersed cells to angiotensin II amide. In the perfused gland, sensitivity of response (lowest effective concentration) to all three stimulants is increased by prior dietary sodium restriction, in contrast to dispersed cells in which increased sensitivity has been reported only to alpha-MSH. The perfused gland is particularly sensitive to angiotensin II amide, and a bolus administration of 1 amol gives significant stimulation in glands from animals on low sodium intake. Electrical (field) stimulation or dopamine administration at 10(-6) mol/l (which is ineffective in dispersed cells) both depress aldosterone secretion by the perfused gland. The data suggest that the sequestered pool of steroid is utilized in the perfused gland for aldosterone secretion. They furthermore suggest that in the intact gland there are mechanisms, which involve neural components, for intraglandular regulation of aldosterone secretion, which are lost in dispersed cells in vitro. Such mechanisms may be involved in sensitivity increases 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.     

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.     

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.     

Evidence for a paracrine role of adrenomedullin in the physiological resetting of aldosterone secretion by rat adrenal zona glomerulosa

Adrenomedullin (ADM) has been recently found to directly inhibit agonist-stimulated aldosterone secretion by dispersed zona glomerulosa (ZG) cells and to stimulate basal catecholamine release by adrenomedullary fragments. In light of the fact that catecholamines enhance aldosterone secretion acting in a paracrine manner, we have investigated whether these two effects of ADM may interact when the integrity of the adrenal gland is preserved. ADM increased basal aldosterone output by adrenal slices containing a core of adrenal medulla, and the effect was blocked by the beta-adrenoceptor antagonist l-alprenolol. In contrast, ADM evoked a moderate inhibition of K(+)-stimulated aldosterone production, and the blockade was complete in the presence of l-alprenolol. The in vivo bolus injection of ADM did not affect plasma aldosterone concentration (PAC) in rats under basal conditions. Conversely, when rat ZG secretory function was enhanced (by sodium restriction or infusion with angiotensin-II [ANG-II]) or depressed (by sodium loading or infusion with the angiotensin-converting enzyme inhibitor captopril), ADM evoked a sizeable decrease or increase in PAC, respectively. The prolonged infusion with the ADM receptor antagonist ADM(22-52) caused a further enhancement of PAC in sodium-restricted or ANG-II-treated rats, and a further moderate decrease of it in sodium-loaded or captopril-administered animals. RIA showed that ADM plasma concentration did not exceed a concentration of 10(-11) M in any group of animals. Under basal conditions, ADM adrenal content was 1.2-2.0 pmol/g, which may give rise to local concentrations higher than 10(-8) M (i.e. well above the minimal effective ones in vitro). ADM adrenal concentration was markedly increased (from two-fold to three-fold) by both ZG stimulatory and suppressive treatments. Collectively, our findings suggest that in vivo 1) ADM, in addition to directly inhibit aldosterone secretion, may enhance it indirectly by eliciting catecholamine release, the two actions annulling each other under basal conditions; 2) under conditions leading to enhanced aldosterone secretion, the direct inhibitory effect of ADM prevails over the indirect stimulatory one, and the reverse occurs when aldosterone secretion is decreased; and 3) the modulatory action of ADM on the aldosterone secretion has a physiological relevance, endogenous ADM being locally synthesized in adrenals.     

Evidence that corticosterone is not an obligatory intermediate in aldosterone biosynthesis in the rat adrenal

CGS 16949A is a potent inhibitor of aromatase in vitro with an IC50 of 0.03 microM for the inhibition of LH-stimulated estrogen biosynthesis in hamster ovaries. In vivo, CGS 16949A leads to sequelae of estrogen deprivation (e.g. regression of DMBA-induced mammary tumors) without causing adrenal hypertrophy in adult rats. To complement these in vitro and in vivo findings, the effect of CGS 16949A on adrenal steroid biosynthesis in rats was investigated in vitro and in vivo. The surprising finding in vitro was that CGS 16949A inhibited aldosterone biosynthesis (IC50 = 1 microM) at concentrations 100 times lower than those for inhibition of corticosterone biosynthesis (IC50 = 100 microM). Moreover, deoxycorticosterone (DOC) concentrations were elevated at all concentrations of CGS 16949A which inhibited aldosterone synthesis. The classical biosynthetic pathway for aldosterone is DOC----corticosterone----18-OH-corticosterone----aldosterone. Thus inhibition of aldosterone biosynthesis, reflected in DOC accumulation, without affecting corticosterone concentrations, indicates that corticosterone is not an obligatory intermediate in the conversion of DOC to aldosterone in the rat. In vivo, CGS 16949A showed a suppression of plasma aldosterone in ACTH-stimulated male rats at doses which did not significantly affect plasma corticosterone. In conclusion, aldosterone measured both in vitro and in vivo must be derived primarily from a biosynthetic pathway in which corticosterone is not obligatory intermediate.     

Simultaneous determination of intracellular free calcium and aldosterone production in bovine adrenal zona glomerulosa

Angiotensin II (AII) induces an initial rapid but transient rise in [Ca2+]i detected with aequorin in bovine adrenal capsule strips. The rise in [Ca2+]i begins immediately after AII addition, reaches a peak in 30 seconds, and returns to near basal values within 5 minutes. The [Ca2+]i transient is receptor-mediated and its height is dose-dependent. The increase in [Ca2+]i is largely due to the release of Ca2+ from an intracellular pool. The uncorrected peak rise in [Ca2+]i after 1 X 10(-6) M beta-[asp1]-AII stimulation is approximately 3 fold, from 110 nM to 300 nM; the peak rise, corrected for diffusion and nonsynchronous cellular response, is from 110 nM to 1.2 microM. Perifusion of aequorin-loaded strips with beta-[asp1]-AII, an aminopeptidase-resistant analog of AII, allows the simultaneous measurement of [Ca2+]i and aldosterone production rate. Levels of agonist which generate a transient rise in [Ca2+]i also produce a sustained increase in aldosterone production rate, but the two events are temporally separated: the transient rise in [Ca2+]i precedes the increase in aldosterone production rate. However, there is a strong correlation, r = 0.94, between the amplitude of the initial [Ca2+]i transient and the magnitude of the sustained increase in steroid production rate.