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.     

Inhibitory actions of somatostatin on cyclic AMP and aldosterone production in agonist-stimulated adrenal glomerulosa cells

Somatostatin (SRIF) is a potent inhibitor of angiotensin II (AII)-stimulated aldosterone production in rat adrenal glomerulosa cells. This inhibition can be prevented by pretreatment of the cells with pertussis toxin, but little else is known about either the specificity or the biochemical bases of SRIF action in this tissue. We therefore conducted detailed studies of the influence of SRIF on steroidogenesis elicited by AII and the other two physiological stimuli of aldosterone production, K+ and adrenocorticotropic hormone (ACTH), in rat adrenal glomerulosa cells. We also determined the effects of SRIF on cytosolic calcium concentration ([Ca2+]i) and cellular cAMP levels. In these studies, SRIF was found to inhibit the aldosterone responses elicited by low concentrations of all three stimuli, which are believed to promote steroid secretion via discrete but interacting cellular signalling mechanisms. In addition, SRIF consistently lowered cellular cAMP levels in the presence of each of the three agents. However, SRIF caused a small and transient increase rather than a decrease in basal ([Ca2+]i), and had no effect on the subsequent elevation of ([Ca2+]i) by AII and K+. These data indicate that activation of a Gi-like protein by SRIF influences steroid responses to all three major regulators of glomerulosa-cell function, and suggest that basal levels of cAMP play a facilitatory or permissive role in the control of aldosterone production by predominantly calcium-mobilizing regulators of mineralocorticoid secretion.     

Dual effects of dopamine in rat adrenal glomerulosa cells

The effects of dopamine (DA) on cAMP production and aldosterone secretion were compared in freshly isolated cells and in primary cultures of rat adrenal glomerulosa cells. Under isolated conditions, glomerulosa cells exhibited dopamine receptors from DA-1 and DA-2 subclass, whereas in cultured conditions, where cells are very sensitive to their known stimuli, cells only exhibited dopamine receptors from the DA-1 subclass. Moreover, unlike freshly isolated cells, dopamine stimulated both cAMP production and aldosterone secretion in 3-day cultured preparations. These effects were receptor specific since they were completely suppressed by Scherring 23390 (a specific DA-1 antagonist) and were unaffected by a beta-adrenergic antagonist. As in vivo rat adrenal cortex contains DA, we discuss a possible involvement of this neurotransmitter in the regulation of aldosterone secretion.     

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.     

Dissociation of aldosterone and prostaglandin biosynthesis in the rat adrenal glomerulosa

The relationship between aldosterone production and prosta-glandin 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 realease. 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.     

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.     

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.     

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 lipoproteins on aldosterone production by isolated glomerulosa cells

We have studied the effects of human high density and low density lipoproteins on adrenocorticotropin (ACTH)-induced aldosterone production by isolated glomerulosa cells obtained from lipoprotein-deficient rats. ACTH increased basal aldosterone production, and the addition of either lipoprotein fraction further enhanced ACTH-induced aldosterone production. This increase could be shown at physiological lipoprotein-cholesterol concentrations. These data are compatible with a role of both high density and low density lipoproteins in supplying cholesterol to aldosterone-secreting glomerulosa cells.     

Effects of lithium on angiotensin-stimulated phosphatidylinositol turnover and aldosterone production in adrenal glomerulosa cells: a possible causal relationship

Turnover of 32P-labelled phosphatidylinositol (PI) was examined in isolated adrenal glomerulosa cells. Increased incorporation of [32P]phosphate into PI in response to angiotensin II was completely prevented by Li+. A simultaneous accumulation of 32P activity in phosphatidic acid (PA) was also observed. Angiotensin II increased the breakdown of PI despite the presence of Li+. These results suggest that Li is a suitable tool to interrupt the accelerated PI cycle in angiotensin-stimulated cells. Aldosterone production of superfused cells was inhibited by Li+ when the cells were stimulated with angiotensin II. On the other hand, Li+ did not inhibit the aldosterone response of the cells to ACTH, a hormone which acts via cyclic AMP and does not enhance PI turnover in these cells. On the basis of these results, we assume that the inhibitory effect of Li+ on aldosterone production is related to its effect on PI turnover.     

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.     

Cyclic AMP-independent effects of ACTH on glomerulosa cells of the rat adrenal cortex

The aim of the present paper is to point out the complexity of ACTH action in glomerulosa cells of the adrenal cortex. We demonstrate that the increase in cAMP production induced by ACTH is the result of a balance between activation of adenylyl cyclase and direct modulation of a PDE2 phosphodiestease activity, an effect mediated by inhibition of cGMP content. Moreover, Ca²⁺ is essential for cAMP production and aldosterone secretion, but its exact primary action is not clearly determined. We recently described that ACTH activated a chloride channel, via the Ras protein, which can be involved in steroidogenesis. ACTH also increases tyrosine phosphorylation of several proteins. These data, together with those of phospholipase C activation, indicate that ACTH action in the adrenal is complex, and most certainly not limited to cAMP production, in particular for the low concentrations of the hormone.

Some years ago, cAMP was considered to be the unique second messenger of ACTH action; now it becomes more and more evident that ACTH triggers complex signaling pathways using several second messengers in a closely interacting way. The most predominant point is that these signals are observed for low concentrations of ACTH.     

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 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.     

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.     

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.