Further investigations on the atrial natriuretic factor (ANF)-induced inhibition of the growth and steroidogenic capacity of rat adrenal zona glomerulosa in vivo

A prolonged infusion with ANF (20 micrograms/kg/h for 7 days) induced atrophy of zona glomerulosa cells and lowering of basal plasma concentration of aldosterone in rats whose hypothalamo-hypophyseal-adrenal axis and renin-angiotensin system had been interrupted by the simultaneous administration of dexamethasone/captopril and maintenance doses of ACTH/angiotensin II. Chronic ANF treatment also caused comparable reductions in the aldosterone response of zona glomerulosa cells to the acute stimulation with angiotensin II, potassium and ACTH. These data are interpreted to indicate that ANF exerts an inhibitory effect on the growth and secretory activity of rat zona glomerulosa, and that the mechanism underlying this action of ANF does not involve blockade of renin release or ACTH secretion.     

Effect of atrial natriuretic peptide on ACTH, dibutyryl cAMP, angiotensin II and potassium-stimulated aldosterone secretion by rat adrenal glomerulosa cells

We examined the effect of rat atrial natriuretic peptide (ANP) on ACTH, dibutyryl cAMP, angiotensin II and potassium-stimulated aldosterone secretion by dispersed rat adrenal glomerulosa cells. ANP inhibited ACTH, angiotensin II and potassium-stimulated aldosterone secretion with IC50's between 0.15-0.20 nM. Inhibition by 10 nM ANP could not be overcome with higher concentrations of these stimuli. ANP shifted the dibutyryl cAMP dose-response curve slightly to the right but did not blunt the maximal aldosterone secretory response. The sites of ANP inhibition in the aldosterone biosynthetic pathway for these stimuli were also examined. ANP inhibited activation of the cholesterol desmolase (CD) enzyme complex by ACTH, angiotensin II and potassium. Activation of the corticosterone methyl oxidase (CMO) enzyme complex by potassium was inhibited by ANP, however, activation by ACTH was not blocked. We concluded that: 1) ANP is a potent inhibitor of ACTH, angiotensin II and potassium-stimulated aldosterone secretion; 2) inhibition of ACTH stimulation is primarily due to lower cAMP levels and; 3) inhibition of angiotensin II and potassium stimulation reflects a block in the activating mechanism of the CMO and/or CD enzyme complexes, whereas CD but not CMO activation by ACTH is inhibited by ANP.     

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 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.     

Atrial natriuretic peptide inhibits the stimulation of aldosterone secretion by ACTH in vitro and in vivo

Previous studies have shown that atrial natriuretic peptide (ANP) inhibits the secretion of aldosterone by isolated adrenal glomerulosa cells stimulated by angiotensin II, ACTH and potassium in vitro and by angiotensin II in conscious unrestrained rats. In this study we investigated further the effects of synthetic ANP on the dose-response curve of aldosterone secretion stimulated by ACTH in vitro. ANP displaced the dose-response curve of aldosterone to ACTH to the right with a significant change in EC50. A similar effect of ANP was reproduced in vivo in conscious unrestrained rats. There was no significant effect of ANP on the corticosterone response to ACTH in vivo. ANP is a potent regulator of aldosterone secretion which may modulate the effects of ACTH on the adrenal in vitro and in vivo.     

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.     

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.     

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.     

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.     

Atrial natriuretic factor (ANF) inhibits the growth and the secretory activity of rat adrenal zona glomerulosa in vivo

A prolonged infusion with ANF induced atrophy of zona glomerulosa cells of rat adrenals and lowering of plasma concentration of aldosterone, without provoking significant changes in PRA. It also notably reduced the rise in the aldosterone plasma level caused by the acute stimulation with angiotensin II. Zona fasciculata cells and the blood concentration of corticosterone did not display any significant change. These findings are interpreted to indicate that ANF exerts an inhibitory effect on the growth and secretory activity of rat zona glomerulosa.     

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.     

Actions of synthetic atrial natriuretic factor on bovine adrenal glomerulosa

Synthetic atrial natriuretic factor (ANF) inhibited aldosterone production by suspensions of bovine adrenal glomerulosa cells. Inhibition by ANF was most pronounced when basal aldosterone production was measured. The effects of angiotensin II (AII), N6,O2'-dibutyryl-adenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP), and elevated potassium were also inhibited by ANF. Inhibition could be partially overcome by high doses of agonist. Inhibition was localized to the early pathway of aldosteronogenesis, to a step before cholesterol side-chain cleavage. ANF had no effect on binding of AII to receptors, on the stimulation by AII of phospholipid turnover, or on the alteration by AII of calcium fluxes.     

Evidence that prolonged alpha-MSH infusion enhances the steroidogenic capacity of rat adrenal zona glomerulosa in vivo

Prolonged infusion with 120 micrograms/kg/day alpha-MSH significantly increased basal plasma level of aldosterone in the rat, as well as raised the acute aldosterone response to a bolus administration of a high dose of ACTH or angiotensin II. These findings suggest that chronic alpha-MSH treatment stimulates the steroidogenic capacity of rat zona glomerulosa.     

Aldosterone production after short-term culture of rat adrenal capsule: responsiveness to angiotensin II, potassium and ACTH

In vitro studies of aldosterone production have traditionally used freshly isolated adrenal glomerulosa tissue. In the present study we examined the effects of short-term culture of rat adrenal capsule on its secretory capacity by measuring both basal and stimulated aldosterone production. Capsules were maintained in culture for 24 h, and then responses to administered angiotensin II (1 X 10(-7) M), potassium (an increase of 2mM) and ACTH (1 X 10(-8) M) were determined during perifusion. Results were compared with responses by freshly isolated adrenal capsule. Although short-term culture reduced basal aldosterone production, responsiveness to administered stimuli was intact and often was greater than that observed with fresh capsular tissue. The results indicate that short-term culture of zona glomerulosa provides a suitable in vitro preparation for examining aldosterone secretory responsiveness to stimuli.     

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.     

Atrial natriuretic factor mRNA and binding sites in the adrenal gland.

The factor inhibiting aldosterone secretion produced by the adrenal medulla may be atrial natriuretic factor (ANF), since the latter abolishes aldosterone release in response to a number of secretagogues, including angiotensin II and K+. In this study we have shown that cells in the adrenal medulla contain ANF mRNA and therefore have the potential to synthesize this peptide. The presence of binding sites for ANF predominantly in the adrenal zona glomerulosa suggests that, if ANF is synthesized in the medulla and transferred to the cortex, it may affect mineralocorticoid status.     

Proopiomelanocortin-derived peptides, phosphoinositides, cAMP, and aldosterone secretion

Since the intracellular messengers of various proopiomelanocortin-derived peptides remain ambiguous at best, we have investigated the possible involvement of phosphoinositide metabolism in aldosterone secretion evoked by alpha-MSH, beta-LPH, as well as ACTH in rat and calf adrenal glomerulosa cells. We have also examined the cAMP responses in the adrenal glomerulosa cells to alpha-MSH comparing it with those of ACTH. Our results showed that neither alpha-MSH, beta-LPH, nor ACTH increased inositol triphosphate (IP3) or other inositol phosphates in adrenal glomerulosa cells while increasing aldosterone secretion from the same cells. Angiotensin II, known to cause hydrolysis of the phosphoinositides, increased IP3 in these adrenal cells in a dose-dependent manner. Both ACTH and alpha-MSH raised the cAMP levels in the calf adrenal glomerulosa cells, although the magnitude of the increase of cAMP in response to ACTH was greater. These findings suggest that IP3 as a mediator of alpha-MSH- and beta-LPH-induced aldosterone secretion is not likely and other mediator(s) may be involved.     

Effects of estradiol on aldosterone secretion in ovariectomized rats

The effects and action mechanisms of estradiol on aldosterone secretion in female rats were studied. Replacement of estradiol benzoate (EB) increased the levels of plasma estradiol and aldosterone in ovariectomized (Ovx) rats. The aldosterone release from zona glomerulosa (ZG) cells was higher in EB-treated rats than in oil-treated animals. EB treatment potentiated the responses of aldosterone release to adrenocorticotropic hormone (ACTH), forskolin (FSK), and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Administration of EB in vivo did not alter cAMP production in response to ACTH or FSK. Although angiotensin II (Ang II) increased aldosterone secretion by rat ZG cells, the stimulatory effect of Ang II on the release of aldosterone was not altered by EB treatment. The conversions of [3H]-deoxycorticosterone to [3H]-corticosterone and [3H]-corticosterone to [3H]-aldosterone in EB-treated groups were greater than those in the oil-treated group. These results suggest that estradiol increases aldosterone secretion in part through the mechanisms involving the activation of the post-cAMP pathway, 11beta-hydroxylase and aldosterone synthase activity.     

Angiotensin II negatively modulates L-type calcium channels through a pertussis toxin-sensitive G protein in adrenal glomerulosa cells.

In bovine adrenal glomerulosa cells, angiotensin II and extracellular K+ stimulate aldosterone secretion in a calcium-dependent manner. In these cells, physiological concentrations of extracellular potassium activate both T-type (low threshold) and L-type (high threshold) voltage-operated calcium channels. Paradoxically, the cytosolic calcium response to 9 mM K+ is inhibited by angiotensin II. Because K+-induced calcium changes observed in the cytosol are almost exclusively due to L-type channel activity, we therefore studied the mechanisms of L-type channel regulation by angiotensin II. Using the patch-clamp method in its perforated patch configuration, we observed a marked inhibition (by 63%) of L-type barium currents in response to angiotensin II. This effect of the hormone was completely prevented by losartan, a specific antagonist of the AT1 receptor subtype. Moreover, this inhibition was strongly reduced when the cells were previously treated for 1 night with pertussis toxin. An effect of pertussis toxin was also observed on the modulation by angiotensin II of the K+ (9 mM)-induced cytosolic calcium response in fura-2-loaded cells, as well as on the angiotensin II-induced aldosterone secretion, at both low (3 mM) and high (9 mM) K+ concentrations. Finally, the expression of both Go and Gi proteins in bovine glomerulosa cells was detected by immunoblotting. Altogether, these results strongly suggest that in bovine glomerulosa cells, a pertussis toxin-sensitive G protein is involved in the inhibition of L-type channel activity induced by angiotensin II.     

Des-Asp-1-angiotensin II. possible role in mediating the renin--angiotensin response in the rat.

Angiotensin II and its heptapeptide fragment, Des-Asp-1-angiotensin II, produced a striking increase in aldosterone secretion in rats pretreated with dexamethasone and morphine to reduce ACTH release. 1-Sar-8-Ala-angiotensin II (10 mug/kg min-1) given simultaneously with angiotensin II (1 mug/min) blocked the aldosterone response to angiotensin II in rats pretreated to reduce ACTH release. In contrast, 1-Sar-8-Ala-angiotensin II at the same dose failed to block the steroid response to Des-Asp-1-angiotensin II (1 mug/min) but a larger dose of 50 mug/kg min-1 of the angiotensin II antagonist blocked completely both the aldosterone and the corticosterone responses to 1 mug/min of Des-Asp-1-angiotensin II. From these data it is suggested that the heptapeptide has a higher affinity for zona glomerulosa receptors than the octapeptide and that Des-Asp-1-angiotensin II mediates, at least in part, the steroidogenic response to the renin-angiotensin system in the rat. The pressor response to Des-Asp-1-angiotensin II was approximately 50% of that produced by the octapeptide in the rat, and 1-Sar-8-Ala-angiotensin II was as effective in partially blocking the pressor response to the octapeptide as in inhibiting the heptapeptide. The present observations indicate a dissociation of adrenal cortex and peripheral arteriolar receptors in their affinity for angiotensin.