Temporal patterns of protein phosphorylation after angiotensin II, A23187 and/or 12-O-tetradecanoylphorbol 13-acetate in adrenal glomerulosa cells.

The temporal patterns of protein phosphorylation in the adrenal glomerulosa cell were analysed by two-dimensional electrophoresis after stimulation with 10 nM-angiotensin II or various agents [10 nM-12-O-tetradecanoylphorbol 13-acetate (TPA), 50 nM-A23187, 1 microM-nitrendipine], administered singly or in combination. These patterns were compared with the temporal patterns of aldosterone secretion induced by the same agonists and antagonists. After 1 and 30 min of stimulation with angiotensin II, different patterns of protein phosphorylation were observed. A comparison of these patterns reveals that: the phosphorylation of only one protein was persistently enhanced during the continuous incubation with angiotensin II; the phosphorylation of five proteins was transiently enhanced (at 1 min but not 30 min); and the phosphorylation of three proteins did not occur at 1 min but was seen at 30 min. Addition of the phorbol ester TPA alone, which at 30 min is without effect in enhancing aldosterone production, has no effect on protein phosphorylation. The combined addition of TPA and the Ca2+ ionophore, A23187, which, like angiotensin II, evokes a sustained increase in aldosterone production, reproduced the temporal patterns of protein phosphorylation seen after angiotensin II action. Manipulations (A23187 alone, angiotensin II plus nitrendipine) which evoke only a transient rise in aldosterone production rate induce a transient rise in cellular protein phosphorylation. The 1 min patterns of phosphorylation seen after A23187 or combined angiotensin II and nitrendipine (a Ca2+ channel antagonist) are similar to those observed after 1 min of angiotensin II stimulation. These results suggest that, when angiotensin II acts, the initial cellular response is mediated by a different mechanism than that responsible for the sustained response.     

Adenosine 3′:5′-cyclic monophosphate production and steroidogenesis by isolated rat adrenal glomerulosa cells. Effects of angiotensin II and [Sar1,Ala8]angiotensin II

Angiotensin II effects on cyclic AMP production and steroid output were studied in a sensitive preparation of isolated rat adrenal glomerulosa cells. With increasing concentrations of angiotensin II logarithmic dose-response curves for aldosterone and cyclic AMP production were similar. The minimum effective dose (0.2nm) for stimulation of aldosterone production also significantly (P<0.001) increased cyclic AMP output. For both aldosterone and cyclic AMP production, the peptide hormone concentration eliciting maximal response (0.2mum) and the ED(50) (median effective dose) values (1nm) were the same; this is consistent with cyclic AMP acting as an intracellular mediator for angiotensin II-stimulated aldosterone production by glomerulosa cells. The angiotensin II antagonist [Sar(1),Ala(8)]angiotensin II inhibited angiotensin II-stimulated corticosterone and aldosterone production in these cells. An equimolar concentration of antagonist halved the response to 20nm-angiotensin II, and complete inhibition was observed with 0.2mum-antagonist. In contrast, [Sar(1),Ala(8)]angiotensin II had no effect on maximally stimulated steroidogenesis induced by serotonin and a raised extracellular K(+) concentration. Increasing concentrations of [Sar(1),Ala(8)]angiotensin II alone decreased corticosterone and aldosterone outputs significantly (P<0.05) at concentrations of 20nm and 2nm of antagonist respectively. A significant (P<0.001) decrease in cyclic AMP production occurred with 2mum antagonist and this was comparable with the decrease in aldosterone production. It is concluded that [Sar(1),Ala(8)]angiotensin II can independently affect glomerulosa-cell steroidogenesis, possibly by modulating adenylate cyclase activity.     

ACTH-induced inhibition of the action of angiotensin II in bovine zona glomerulosa cells. A modulatory effect of cyclic AMP on the angiotensin II receptor.

Both angiotensin II and adrenocorticotropic hormone (ACTH) are well known to play a crucial role on the regulation of aldosterone production in adrenal glomerulosa cells. Recent observations suggest that the steroidogenic action of ACTH is mediated via the cAMP messenger system, whereas angiotensin II acts mainly through the phosphoinositide pathway. However, there have been no reports concerning the interaction between the cAMP messenger system activated by ACTH and the Ca2+ messenger system induced by angiotensin II. Both ACTH and angiotensin II simultaneously act on adrenal cells for regulating steroidogenesis under physiological conditions. Thus the present experiments were performed to examine the effect of ACTH on the action of angiotensin II by measuring angiotensin II receptor activity, cytosolic Ca2+ movement, and aldosterone production. The major findings of the present study are that short-term exposure to a high dose of ACTH (10(-7) M) inhibited 125I-angiotensin II binding to bovine adrenal glomerulosa cells, decreased the initial spike phase of [Ca2+]i induced by angiotensin II, and inhibition of angiotensin II-induced aldosterone production. Low dose of ACTH (10(-10) M), which did not increase cAMP formation, did not affect angiotensin II receptor activity. These studies have shown that angiotensin II receptors of bovine adrenal glomerulosa cells can be down-regulated by 1 mM dibutyryl cyclic AMP, as well as by effectors which are able to activate cAMP formation (10(-7) M ACTH and 10(-5) M forskolin). The rapid decrease in angiotensin II receptors induced by 10(-7)M ACTH was associated with a decreased steroidogenic responsiveness and a decreased rise in the [Ca2+]i response induced by angiotensin II. These studies show that the cAMP-dependent processes activated by ACTH have the capacity to interfere with signal transduction mechanisms initiated by receptors for angiotensin II.     

Effect of cyclosporin A on aldosterone production by dispersed rabbit adreno-capsular cells

The effect of cyclosporin A on aldosterone production by dispersed adreno-capsular cells from rabbit was examined. Cyclosporin A significantly stimulated aldosterone production at concentrations of 10(-7) M and 10(-6) M. The maximum stimulation of aldosterone production by cyclosporin A (at 10(-6) M) was comparable to that by angiotensin II at 10(-8) M). This stimulating effect of cyclosporin A on aldosterone production was not accompanied by an increase in cyclic AMP production, and was not inhibited by a calcium-channel blocker, nicardipine. These results suggest that the aldosterone-stimulating action of cyclosporin A at these concentrations is not mediated by a known second messenger system such as channel-linked Ca2+ inflow or cyclic AMP.     

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.     

Magnesium ion: a possible physiological regulator of aldosterone production

We examined the direct effect of magnesium ion on aldosterone production by adrenal cells using collagenase-dispersed zona-glomerulosa cells in rats. The effects of magnesium on aldosterone production stimulated by angiotensin II or ACTH were also investigated. Both magnesium sulphate (MgSO4) and magnesium chloride (MgCl2) (0 to 2 mM) decreased aldosterone production in a dose-dependent manner. In comparison with magnesium-free medium, 2 mM MgSO4 inhibited aldosterone production by 73% and MgCl2 by 65%. In addition, MgSO4 showed an inhibitory effect on aldosterone production stimulated by angiotensin II (10pM to 10nM), whereas it had no significant effect on aldosterone production due to ACTH stimulation (10pM to 10nM). These data suggest that magnesium has an inhibitory action on aldosterone production in vitro and may be a physiological regulator of aldosterone production.     

Insulin prevents glucose induced inhibition of angiotensin II-stimulated aldosterone secretion

In humans with diabetes mellitus or in individuals given infusions of insulin or insulin plus glucose, plasma aldosterone levels have been reported to be suppressed. Whether insulin has a direct effect to suppress aldosterone secretion by the adrenal gland has not been established. The effect of insulin on glucose-induced inhibition of angiotensin II-stimulated aldosterone secretion was examined. The effect of glucose and insulin plus glucose on angiotensin II-stimulated aldosterone secretion was examined in isolated perfused canine adrenal glands. In the absence of insulin, 15.6 mM glucose decreased angiotensin II-stimulated aldosterone secretion by 35 +/- 7%, while in the presence of insulin the same glucose concentration had no significant effect on angiotensin II-stimulated aldosterone secretion. In contrast, insulin had no effect on NaCl-induced inhibition of angiotensin II-stimulated aldosterone secretion. Neither insulin alone nor saline vehicle affected angiotensin II-stimulated aldosterone secretion. These results (1) demonstrate that insulin can prevent inhibition of glucose-induced angiotensin II-stimulated aldosterone secretion, possibly by preventing a glucose-induced decrease in cell volume, and (2) suggest that the suppressed plasma level of aldosterone found in individuals with diabetes mellitus may in part be due to the direct effects of hyperglycemia on the adrenal gland secretion of aldosterone.     

Dopaminergic modulation of aldosterone secretion: effect of sodium balance and postural changes

The influence of an increased endogenous production of angiotensin II and of sodium homeostasis upon the response of plasma aldosterone to metoclopramide administration has been investigated in 5 normal volunteers. Our results show that the increase of plasma aldosterone after metoclopramide administration is independent of angiotensin II, ACTH and potassium, and that it increases even further due to the endogenous production of angiotensin II induced by postural changes. The state of sodium balance seems to influence the response of plasma aldosterone to metoclopramide administration as it occurs with other stimuli of aldosterone secretion.     

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.     

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.     

Angiotensin II and aldosterone regulation

The circulating renin-angiotensin system is a major regulator of the secretion of the adrenocortical hormone, aldosterone. This renin-angiotensin aldosterone system is important in the control of salt and water balance and blood pressure. This review describes the historical background leading to the discovery of aldosterone in the 1950s and the recognition in the 1960s that angiotensin II was involved in its control. Although angiotensin II is important in the regulation of aldosterone secretion, its action is influenced by multiple other factors, especially potassium and atrial natriuretic peptide. In addition to the circulating renin-angiotensin system, a local renin-angiotensin system is present in the zona glomerulosa cell. This local system also appears to be involved in the regulation of aldosterone production. The mechanism by which angiotensin II stimulates the adrenal zona glomerulosa cell is described in some detail. Angiotensin II interacts with the angiotensin receptor (AT1) membrane receptor that is coupled to cellular second messengers. Specific AT1 receptor antagonists are now clinically used to block angiotensin II's action on various target organs, including the adrenal gland.     

Lack of inhibition of ACTH-induced aldosterone stimulation by des-asp1-,ileu8-angiotensin II in man.

In 5 normal men an intravenous injection of 0.5 mg of synthetic 1-24 ACTH caused a significant increase in plasma aldosterone and a simultaneous intravenous infusion of 600 ng/kg/min of des-asp1-, ileu8-angiotensin II (AIIIA) did not inhibit this increase. Since this dose of AIIIA is known to inhibit an angiotensin II-induced increase in plasma aldosterone in normal men, the present results suggest that the ACTH-induced aldosterone stimulation is mediated by an adrenocortical receptor which is different from angiotensin II receptors.     

An examination of possible mechanisms of angiotensin II-stimulated steroidogenesis.

Dog and rat adrenal glomerulosa cells and subcellular fractions have been utilized to evaluate the mechanism of angiotensin II- and angiotensin III-induced aldosterone production. The effects of angiotensin, ACTH, and potassium have been compared on cyclic AMP and cyclic GMP in isolated glomerulosa cells and adenylate cyclase activity in subcellular fractions. The effect of angiotensin II has also been assessed on Na+-K+-activated ATPase of plasma membrane enriched fractions of dog and rat adrenals. We have demonstrated no effect of angiotensin II or angiotensin III on either adenylate cyclase, cyclic AMP, cyclic GMP, or Na+-K+-dependent ATPase activity over a wide range of concentrations. Potassium ion in concentrations that stimulate significant aldosterone production was also without effect. The negative effects of angiotensin and potassium were contrasted against a positive correlation between an ACTH-induced effect on aldosterone production, adenylate cyclase, and cyclic AMP accumulation. These studies have served to demonstrate that neither adenylate cyclase, cyclic AMP, cyclic GMP, or Na+-K+-activated ATPase seem to be directly involved in the mechanism of action of angiotensins on aldosterone production in the rat and dog adrenal glomerulosa.     

Contrasting effects of sn-1,2-dioctanoyl glycerol as compared to other protein kinase C activators in adrenal glomerulosa cells

Angiotensin II acts on adrenal glomerulosa cells to induce the phospholipase C-mediated generation of inositol trisphosphate and sn-1,2-diacylglycerol as the major products of inositol phospholipid breakdown. This last product is known to activate protein kinase C, but its role in the action of angiotensin II on steroidogenesis has not been defined. We report herein that, in bovine adrenal glomerulosa cells, protein kinase C activators, such as phorbol 12,13-dibutyrate, 12-O-tetradecanoylphorbol-13-acetate, mezerein and sn 1,2 oleoyl acetoylglycerol, each failed to increase steroidogenesis. These results contrast with our recent report on the enhancement of aldosterone output by sn-1,2-dioctanoylglycerol (DiC8) [J. Steroid Biochem. 35 (1990) 19-33]. In addition, the difference between DiC8 and the other protein kinase activators was also observed in the pattern of 86Rb efflux from preloaded glomerulosa cells; only DiC8 mimicked the effect of angiotensin II on ion fluxes. Furthermore, staurosporine, a potent inhibitor of protein kinase C, was capable of amplifying the aldosterone output induced by a maximally effective concentration of DiC8 or angiotensin II. These data suggest that the effect of the cell permeant DiC8 on aldosterone biosynthesis either is not mediated by protein kinase C activation, or is mediated by a phorbol ester-insensitive isoenzyme of protein kinase C.     

Steroid production by quartered and capsular rat adrenal gland in response to haemorrhage.

60 min after rapid bleeding (1.5--2.0 per cent of b. w.) both aldosterone and corticosterone production rate by quartered rat adrenals were found to be elevated. However, no difference was observed in the rate of aldosterone and corticosterone production by capsular adrenals of sham operated and hypovolaemic rats. Corticosterone production rate by decapsulated adrenals was much more higher after haemorrhage than in the control group. The same alterations could be observed incubating adrenal tissue with ACTH (0.3 mug per ml). Steroid production rate by quartered adrenals of sodium deficient rats was not affected by high in vitro concentration of angiotensin II (2.5 mug per ml). It is concluded that the effect of acute blood loss on corticosteroid biosynthesis of the rat is mediated by ACTH alone.     

Aldosterone regulation in anephric patients.

The response of plasma aldosterone to hemodialysis, 3 h orthostatism, K-loading and angiotensin II and ACTH infusions has been studied. Hemodialysis, orthostatism and angiotensin II infusion do not modify aldosterone levels. By the contrary ACTH and potassium originate a significant increase in plasma aldosterone. They seem to be the main aldosterone secretion regulators in the absence of renin production.     

Control of calcium homeostasis by angiotensin II in adrenal glomerulosa cells through activation of p38 MAPK

Angiotensin II-induced activation of aldosterone secretion in adrenal glomerulosa cells is mediated by an increase of intracellular calcium. We describe here a new Ca2+-regulatory pathway involving the inhibition by angiotensin II of calcium extrusion through the Na+/Ca2+ exchanger. Caffeine reduced both the angiotensin II-induced calcium signal and aldosterone production in bovine glomerulosa cells. These effects were independent of cAMP or calcium release from intracellular stores. The calcium response to angiotensin II was more sensitive to caffeine than the response to potassium, suggesting that the drug interacts with a pathway specifically elicited by the hormone. In calcium-free medium, calcium returned more rapidly to basal levels after angiotensin II stimulation in the presence of caffeine. Thapsigargin had no effect on these kinetics, but diltiazem, which inhibits the Na+/Ca2+ exchanger, markedly reduced the rate of calcium decrease and abolished caffeine action. The involvement of this exchanger was supported by the effect of cell depolarization and of a reduction of extracellular sodium on the rate of calcium extrusion. We also determined the mechanism of angiotensin II action on the exchanger. Phorbol esters reduced the rate of calcium extrusion, which was increased by baicalein, an inhibitor of lipoxygenases, and by SB 203580, an inhibitor of the p38 MAPK. Finally, we showed that angiotensin II acutely activates, in a caffeine-sensitive manner, p38 MAPK in glomerulosa cells. In conclusion, in bovine glomerulosa cells, the Na+/Ca2+ exchanger plays a crucial role in extruding calcium, and, by reducing its activity, angiotensin II influences the amplitude of the calcium signal. The hormone exerts its action on the exchanger through a caffeine-sensitive pathway involving the p38 MAPK and lipoxygenase products.     

sn-1,2 dioctanoylglycerol mimics the effects of angiotensin II on aldosterone production and potassium permeability in isolated bovine glomerulosa cells

In order to elucidate the possible role in glomerulosa cells of diacylglycerol released by angiotensin II we have studied the action of a synthetic diacylglycerol, sn-1,2-dioctanoylglycerol (DiC8), on aldosterone production and potassium permeability in bovine adrenal cells. DiC8 elicited an increase in 86Rb efflux from cells previously equilibrated with the isotope. The action of DiC8 on the rate coefficient for 86Rb efflux was similar to that previously described for angiotensin II (Am. J. Physiol. 254 (1988) E144-149), i.e. DiC8 induced an immediate increase in 86Rb efflux followed by a sustained decrease in potassium permeability. This DiC8 induced inhibition was observed even in the presence of depolarizing concentrations of potassium. The effect of DiC8 on aldosterone secretion from adrenal glomerulosa cells was measured using a perifusion system. DiC8 (300 microM) caused a significant increase of aldosterone production, comparable to that seen with angiotensin II (100 nM). These results indicate that DiC8 has similar effects to angiotensin II on both potassium permeability and steroidogenesis, which suggests that activation of protein kinase C is involved in the changes of ionic permeability induced by this hormone in bovine adrenal glomerulosa cells.     

Adrenal cortex production of aldosterone "in vitro" caused by angiotensin II: effects of indomethacin

Angiotensin II increases aldosterone production of isolated and superfused bovine adrenal glands. Indomethacin shows a bi-phasic effect on the production of the above-mentioned hormone, inhibitory at low doses (0,2 microgram/ml), stimulatory at high doses (5.0 microgram/ml). Preincubation with this drug impedes the increase of aldosterone production induced by angiotensin II. It is likely that the steroidogenetic effect of angiotnesin II is carried out through the PGs.