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.     

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.     

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 atrial natriuretic peptide receptors in human aldosteronoma

The effect of synthetic alpha-human atrial natriuretic peptide (ANP) on aldosterone secretion was studied in human aldosterone producing adrenocortical adenoma obtained surgically from a patient with primary aldosteronism and in human apparently normal adjacent adrenal cortical tissues obtained from a patient with pheochromocytoma, in vitro. Apparently normal adrenal cortical tissue responded to ANP with the known inhibition of aldosterone secretion. In contrast, the aldosterone producing adenoma did not respond to ANP. When stimulated by either ACTH or angiotensin II, there is no inhibition by ANP in the adenoma tissue, whereas normal tissue was inhibited. Immunohistochemical examination utilizing an ANP-receptor antiserum demonstrated that there was no evidence of binding site in the cortical adenoma, in contrast, zona glomerulosa cells in the cortical tissues adjacent to either aldosterone producing adenoma or pheochromocytoma were densely stained. This apparent lack of ANP-receptors is an associated finding with the hypersecretion of aldosterone in the aldosterone producing adenoma.     

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.     

Effect of metoclopramide on adrenal secretion in sheep: influence of dexamethasone and sodium intake

Metoclopramide, a competitive dopamine antagonist, stimulates aldosterone in man and monkey without affecting cortisol secretion. In sheep, metoclopramide also stimulates aldosterone but ist action on adrenocortical secretion is more controversial. To clarify the action of metoclopramide in conscious sheep, the response of plasma aldosterone, cortisol, angiotensin II and potassium were studied after 0.16 and 0.64 mg/kg metoclopramide, with and without pretreatment with dexamethasone. The effect of sodium status on the response was also studied by repeating the experiments after 7 days of dietary sodium restriction. In the absence of dexamethasone, plasma aldosterone was significantly increased by metoclopramide in both sodium-replete and restricted sheep. In sodium-replete sheep, plasma cortisol was also increased by 0.64 mg/kg, and by both doses when salt-restricted. However all cortisol responses were completely suppressed by dexamethasone pretreatment. Dexamethasone also suppressed the aldosterone response to metoclopramide in sodium-replete but not in sodium-restricted sheep where significant responses of aldosterone to both doses of metoclopramide still occurred without changes in plasma angiotensin II or potassium. While a nonspecific stress effect of metoclopramide can contribute to the aldosterone response, these results show that the sheep's adrenal glomerulosa is capable of responding to metoclopramide without change in ACTH, angiotensin or potassium.     

Atrial and brain natriuretic peptide in adrenal steroidogenesis.

We elucidated the role of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human and bovine adrenocortical steroidogenesis. The urinary volume, sodium excretion and cyclic GMP (cGMP) excretion and plasma cGMP were markedly increased by the synthetic alpha-human ANP (alpha-hANP) infusion in healthy volunteers. Plasma arginine vasopressin (AVP) and aldosterone levels were significantly suppressed. Both ANP and BNP inhibited aldosterone, 19-OH-androstenedione, cortisol and DHEA secretion dose-dependently and increased the accumulation of intracellular cGMP in cultured human and bovine adrenal cells. alpha-hANP significantly suppressed P450scc-mRNA in cultured bovine adrenal cells stimulated by ACTH. Autoradiography and affinity labeling of [125I]hANP, and Scatchard plot demonstrated a specific ANP receptor in bovine and human adrenal glands. Purified ANP receptor from bovine adrenal glands identified two distinct types of ANP receptors, one is biologically active, the other is silent. A specific BNP receptor was also identified on the human and bovine adrenocortical cell membranes. The binding sites were displaced by unlabelled ANP as well as BNP. BNP showed an effect possibly via a receptor which may be shared with ANP. The mean basal plasma alpha-hANP level was 25 +/- 5 pg/ml in young men. We confirmed the presence of ANP and BNP in bovine and porcine adrenal medulla. Plasma or medullary ANP or BNP may directly modulate the adrenocortical steroidogenesis. We demonstrated that the lack of inhibitory effect of alpha-hANP on cultured aldosterone-producing adenoma (APA) cells was due to the decrease of ANP-specific receptor, which caused the loss of suppression of aldosterone and an increase in intracellular cGMP.     

Effect of metoclopramide on angiotensins, aldosterone, and atrial peptide during hypoxia

The coupling of aldosterone with renin is altered during acute hypoxemia. We measured the various components of the renin-angiotensin system and the plasma levels of immunoreactive atrial natriuretic factor (iANF) during room air and hypoxic gas-mixture breathing before and after administration of metoclopramide, a competitive antagonist of dopamine. Seven resting volunteers were studied 1 wk apart under room air and hypoxic conditions (inspired O2 fraction 0.12). During hypoxemia, the release of aldosterone induced by metoclopramide was significantly smaller. This change was associated with a slight increase in iANF and with a decrease in plasma angiotensin II levels, without any change in immunoreactive blood angiotensin I concentrations. Plasma electrolytes and blood acid-base status did not show relevant changes, nor did blood pressure and heart rate. We conclude that the decreased aldosterone concentrations seen under hypoxemia are related to decreased angiotensin II levels. Other influences, such as elevated ANF, may also mediate this effect.     

Alpha-human atrial natriuretic polypeptide inhibits 19-hydroxy-androstenedione secretion by human adrenal cells

We investigated the effect of ACTH, angiotensin II (AII), and alpha-human atrial natriuretic polypeptide (alpha-hANP) which plays an important role of water-electrolytes balance, on 19-hydroxyandrostenedione (19-hydroxyandrost-4-ene-3,17-dione, 19-OH-A-dione) secretion by cultured human adrenal cells. 19-OH-A-dione in culture media was measured using a specific RIA. Basal 19-OH-A-dione secretion by adrenal cells was 0.69 +/- 0.08 ng/3h/10(6) cells and significantly rose to 1.17 +/- 0.14 ng/3h/10(6) cells in the presence of ACTH, but not in the presence of A II. These results demonstrate that 196-OH-A-dione is directly secreted from adrenal cells. alpha-hANP significantly inhibited both basal and ACTH-stimulated 19-OH-A-dione secretions, as well as aldosterone. These results demonstrate that alpha-hANP inhibits aldosterone activity by means of the inhibition of both aldosterone and 19-OH-A-dione (an aldosterone amplifier) secretion by adrenal cells.     

Comparative in vitro responses of fetal, pregnant and non-pregnant rabbits' adrenal glands to steroidogenic agents

The in vitro secretion of aldosterone and corticosterone by the adrenal glands of fetal (day 30), pregnant and non-pregnant rabbits was examined under basal and stimulated conditions. In general, non-pregnant animals basally secreted less aldosterone than either pregnant or fetal rabbits, whereas basal corticosterone secretion by pregnant animals exceeded that of either fetal or non-pregnant animals. At similar doses of adrenocorticotropin (ACTH), fetal and pregnant adrenal glands produced comparatively more aldosterone than non-pregnant animals, while corticosterone secretion was accelerated to a greater degree in fetal rabbits than in the other groups. Angiotensin II had its greatest effect on the aldosterone secretory rates of fetal and non-pregnant animals without affecting corticosterone secretion in any group. Elevated potassium (K+) enhanced the secretory rates of aldosterone and corticosterone in fetal animals, while increasing only aldosterone secretion in non-pregnant rabbits. Serotonin accelerated aldosterone secretion in all animals, whereas it increased corticosterone secretion only in non-pregnant animals. These results suggest that (1) in fetal rabbits, the secretory rates of both aldosterone and corticosterone are regulated primarily by ACTH and to a much lesser extent by angiotensin II and K+, (2) the corticosterone secretory rates of pregnant and non-pregnant rabbits are controlled mainly by ACTH, and (3) aldosterone secretion by non-pregnant animals is regulated primarily by angiotensin II and secondarily by ACTH and K+, while in pregnant animals ACTH may be the primary regulator of aldosterone secretion as it is in the fetus.     

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.     

Cyclic GMP Regulates Nicotine-Induced Secretion from Cultured Bovine Adrenal Chromaffin Cells: Effects of 8–Bromo–Cyclic GMP, Atrial Natriuretic Peptide, and Nitroprusside (Nitric Oxide)

Methacholine, atrial natriuretic peptide (ANP), nitroprusside (nitric oxide), angiotensin II, and bradykinin raised cyclic GMP (cGMP) levels in cultured bovine adrenal chromaffin cells. The role of cGMP in secretion from chromaffin cells was examined using 8-bromo-cGMP. This analogue had no effect on basal secretion or secretion due to angiotensin II, bradykinin, or a high K+ level but potentiated secretion due to low doses of nicotine. At supramaximal doses of nicotine, 8-bromo-cGMP inhibited secretion. These effects of 8-bromo-cGMP were not due to changes in the nicotine-induced rise in cytosolic calcium concentration. A potentiation of secretion due to low doses of nicotine was also found following simultaneous addition of ANP or nitroprusside, a result suggesting that ANP and nitric oxide (endothelium-derived relaxing factor) could be important regulators of secretion from adrenal chromaffin cells.     

Alpha-human natriuretic polypeptide (alpha-hANP) specific binding sites in bovine adrenal gland

The effects of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) on steroidogenesis in bovine adrenocortical cells in primary monolayer culture were investigated. alpha-hANP did not inhibit basal aldosterone secretion. alpha-hANP induced a significant dose-dependent inhibition of basal levels of cortisol and dehydroepiandrosterone (DHEA) secretion and also of ACTH (10(-8) M)-stimulated increases in aldosterone, cortisol and DHEA secretion. Visualization of [125I]alpha-hANP binding sites in bovine adrenal gland by an in vitro autoradiographic technique demonstrated that these sites were highly localized in the adrenal cortex, especially the zona glomerulosa. These results suggest that the adrenal cortex may be a target organ for direct receptor-mediated actions of alpha-hANP.     

Effect of verapamil on the aldosterone-stimulating properties of metoclopramide: in vitro and in vivo studies

The role of calcium in the regulation of aldosterone secretion has been recently clarified. Angiotensin II and potassium stimulate aldosterone secretion through a calcium-entry dependent mechanism, while ACTH action is both calcium and cyclic AMP dependent. To establish whether also the so-called aldosterone dopaminergic regulatory system is calcium-dependent we have studied, in vitro and in vivo, the effect of verapamil, a calcium entry blocker agent, on the aldosterone-stimulating properties of the antidopaminergic drug, metoclopramide. In the rat adrenal cells perfusion system, verapamil blocked both angiotensin II and metoclopramide-stimulated aldosterone. This effect on metoclopramide action seems to be present also in vivo in normal subjects: in fact aldosterone response was slightly but significantly reduced after pretreatment with verapamil. In conclusion the results suggest that also the dopaminergic system could regulate aldosterone secretion through calcium-mediated mechanisms.     

Porcine brain natriuretic peptide, another modulator of bovine adrenocortical steroidogenesis

Porcine brain natriuretic peptide (pBNP) significantly inhibited aldosterone production stimulated by an angiotensin II analog and ACTH-stimulated cortisol secretion, together with simultaneously increasing the formation of cGMP in dispersed bovine adrenocortical cells. Receptors for pBNP were identified in bovine adrenal gland using an in vitro receptor autoradiographic technique and studies of 125I-pBNP binding. In vitro receptor autoradiography demonstrated specific binding sites for 125I-pBNP in bovine adrenal cortex. Complete displacement of 125I-pBNP by unlabeled pBNP or human atrial natriuretic peptide (hANP) can take place at these sites. Analysis of 125I-pBNP binding to bovine adrenocortical membrane fractions showed that the adrenal cortex had high-affinity, low-capacity pBNP-binding sites, with a dissociation constant (Kd) of 2.32 +/- 0.33 x 10(-10) M (mean +/- SE) and a maximal binding capacity (Bmax) of 36.7 +/- 1.6 fmol/mg protein. Moreover, the specific binding sites for 125I-pBNP were completely displaced not only by unlabeled pBNP but also by unlabeled hANP. The hANP dose required for 50% inhibition of specific 125I-pBNP binding was almost identical to that for pBNP (IC50 values for hANP and pBNP: 8.5 x 10(-10) and 6.5 x 10(-10) M, respectively). These results suggest that pBNP exerts a suppressive effect on bovine adrenocortical steroidogenesis via a receptor which may be shared with ANP.     

In vitro responsiveness of aldosterone-producing adenoma to angiotensin II, K and ACTH

In vitro responsiveness to various stimulators of aldosterone secretion was studied in a perifusion system using slices obtained from three aldosterone-producing adenomas (APAs), three adjacent nontumorous glands and three normal adrenal glands. All three APA tissues responded to angiotensin II, K and ACTH in vitro. Angiotensin II (10 nM), K (12 mM) and ACTH (10 nM) caused more than a 2-fold increase in aldosterone secretion. The sensitivity of APA tissues to angiotensin II was identical to that in normal adrenal cortex. In slices obtained from APA, angiotensin II induced rapid increases in [3H]inositol and [45Ca] efflux, both of which preceded the aldosterone response. These results suggest that APA cells have an almost normal transducing system to stimulators of aldosterone secretion.     

Metoclopramide fails to stimulate aldosterone secretion and inhibits serotonin-mediated aldosterone secretion in the rat

The acute and chronic effects of metoclopramide on aldosterone secretion in the rat model were examined. Metoclopramide 50 micrograms iv in dexamethasone-treated rats did not increase plasma aldosterone concentration. Chronic infusion of metoclopramide (72 micrograms/hr) over 5 days also did not show any increase in the plasma or urinary aldosterone concentration when compared with control rats. Metoclopramide in vitro showed no effect on aldosterone secretion from rat adrenal capsular cells but it inhibited serotonin-mediated aldosterone secretion from the same cells significantly.     

Bovine adrenal chromaffin granules are a site of synthesis of atrial natriuretic factor

We have previously reported the existence of a peptide factor in the adrenal medulla which inhibits aldosterone secretion in cultured bovine zona glomerulosa cells. The acid extracts of chromaffin granules from bovine adrenal medulla were purified by a four step high performance liquid chromatography procedure. Two active fractions exhibited sequence homology with bovine atrial natriuretic factor ANF (Ser99-Tyr126) and its polypeptide precursor (Asn1-Tyr126). The occurrence of both precursor and mature forms of ANF within chromaffin granules indicates the endogenous character of ANF in the adrenal medulla and suggests the potential usefulness of cultured adrenal chromaffin cells for investigating the synthesis, maturation and secretion of atrial peptides.     

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.     

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.