Primary aldosteronism (PA) was first described in patients with unilateral aldosterone-producing adenomas (1). It is characterized by increased aldosterone secretion causing salt retention, increased urinary potassium excretion, relative hypervolemia, suppressed plasma renin activity (PRA), and hypertension. PA is the most common curable form of secondary hypertension as it affects 4.3% of the general hypertensive population, 9.5% of patients referred to hypertension clinics (2), and up to 20% of those with resistant hypertension (3). PA is most often secondary to bilateral adrenal hyperplasia (BAH; 50–70% of cases) or to an aldosterone-producing adenoma (APA; 30–50% of cases) (4). The classical concept that a unique unilateral aldosteronoma is the causative lesion responsible for a high proportion of this surgically curable form of PA was recently challenged by the identification of zona glomerulosa (ZG) hyperplasia and nodulation adjacent to aldosteronomas when resected adrenals are examined carefully (5–7).

In order to prevent cardiovascular, metabolic and renal morbidities, early diagnosis and management of PA are mandatory (8–10). Unilateral adrenalectomy (UA) provides superior benefit compared to medical therapy in lateralized PA in terms of cardiovascular outcomes (11, 12), quality of life (12), and all-cause mortality (13); however, in BAH, pharmacological blockade of aldosterone excess using mineralocorticoid receptor antagonists, such as spironolactone or eplerenone, is the recommended treatment (4). Therefore, subtyping of PA is required to direct patients to surgical vs. medical therapy (4). To date, adrenal vein sampling (AVS) is the gold standard to differentiate lateralized from bilateral sources of PA (4) because adrenal imaging provides poor specificity in detecting lateralized cases (14) except in patients <35 years old (15).

In PA excess, plasma aldosterone concentration (PAC), despite suppressed renin activity, is not really autonomous, as frequently stated. It could be autonomous if it was solely or constitutively regulated by somatic and germline mutations of various ion channels genes regulating intracellular ionic homeostasis and cell membrane potential as reviewed elsewhere (16). In fact, several autocrine/paracrine hormones and regulatory mechanisms (17) activate variable levels of aberrant eutopic or ectopic receptors (18), which regulate aldosterone secretion either in unilateral adenomas or in BAH. In this review, we will focus on the role of one of these hormones, the adrenocorticotropic hormone (ACTH), in stimulating aldosterone secretion in normal and pathologic conditions and briefly mention others which play similar roles in PA.

Renin is an enzyme produced primarily by the juxtaglomerular apparatus of the kidney and its release is the rate-limiting step in the regulation of the RAS (19, 20); it is controlled by four factors: (1) the macula densa comprises chemoreceptors for monitoring the sodium and chloride loads present in the distal tubule, (2) juxtaglomerular cells acting as pressure transducers that sense stretch of the afferent arteriolar wall and thus renal perfusion pressure, (3) the sympathetic nervous system (SNS), which increases the release of renin, particularly in response to upright posture, in addition to (4) inhibiting factors, including K⁺, Ca⁺⁺, angiotensin II, and atrial natriuretic peptides (19).

The action of renin on angiotensinogen, synthesized in the liver, generates angiotensin I (19). Angiotensin-converting enzyme (ACE), localized in cell membranes particularly of the lung, cleaves angiotensin I into angiotensin II, which is the main biologically active angiotensin (19). Angiotensin II functions through the AT-1 receptor (AT1R) to maintain normal extracellular volume and blood pressure by increasing aldosterone secretion from the ZG via increased transcription of CYP11B2 (aldosterone synthase) (Figure 1) as well as constricting vascular smooth muscle, releasing norepinephrine, and epinephrine from the adrenal medulla, enhancing the activity of the SNS and finally promoting the release of vasopressin (19).

Zona glomerulosa cells are organized in rosette structures that spontaneously generate periodic depolarizing changes in membrane potential that are modulated in frequency by angiotensin II and extracellular K⁺ (21, 22); Angiotensin II induces cell membrane depolarization most probably due to a Gi-mediated shift in the voltage dependence of channel activation toward more negative potentials thereby increasing intracellular Ca²⁺ signal, which stimulates hormone-sensitive lipase and steroidogenic acute regulatory protein (StAR). Another mechanism by which angiotensin II binding to AT1R stimulates aldosterone secretion implicates activating the phospholipase C/inositol 1,4,5-trisphosphate pathway, releasing Ca²⁺ stores from the endoplasmic reticulum, and activation of T-type voltage-gated Ca²⁺ channels (23) (Figure 1).

Dopamine, atrial natriuretic peptide, and heparin inhibit aldosterone secretion. The secretion of aldosterone is restricted to the ZG because of zone-specific expression of aldosterone synthase (CYP11B2), which is regulated by the activation of calcium signaling (24).

The binding of free aldosterone to the mineralocorticoid receptor in the cytosol of epithelial cells (24), principally in the kidney, controls potassium homeostasis and maintains normal intravascular volume by increasing intestinal and renal Na⁺ and Cl⁻ absorption and reabsorption, respectively. Increased production of aldosterone in PA results in sodium retention, hypertension, and can also result in hypokalemia (20). In addition to the two most common subtypes of PA (BAH in 50–70% of the cases and APA in 30–50%), less frequent causes include primary (unilateral) adrenal hyperplasia (5%), aldosterone-producing adrenocortical carcinoma (<1%), familial hyperaldosteronism (1%), and ectopic aldosterone-producing adenoma or carcinoma (<0.1%). The mechanisms implicated in the pathophysiology of PA are not fully elucidated. Somatic and germline mutations in ion channels genes regulating intracellular ionic homeostasis and cell membrane potential were described in sporadic APA and type-III familial PA (25–28) (Figure 1). Somatic mutations in the potassium channel gene KCNJ5 are found in almost 30–40% of aldosteronomas and alter channel selectivity allowing enhanced Na⁺ conductance. Na⁺ influx results in cell depolarization, the activation of voltage-gated Ca²⁺ channels, aldosterone production, and cell proliferation (25, 29). Somatic and germline mutations in CACNA1D gene encoding a voltage-gated calcium channel result in channel activation and less depolarized potentials causing increased Ca²⁺ influx, aldosterone production and cell proliferation in affected ZG cells (27, 30). Mutations in ATP1A1 gene (encoding the Na⁺/K⁺ ATPase α subunit) and ATP2B3 gene (encoding the plasma membrane Ca²⁺ ATPase) were identified in 5.2 and 1.6%, respectively of patients in a series of APA (26). Mutations in CACNA1H gene, which encodes a voltage-gated calcium channel (Cav3.2) were discovered in children with PA; they result in impaired channel inactivation and activation at more hyperpolarized potentials, producing increased intracellular Ca²⁺ and aldosterone excess (31). Different mutations in the genes described above are found in different aldosterone-producing nodules from the same adrenal, suggesting that somatic mutations are independent events (32, 33).

No mutations of any of the above ion channel genes were found in BAH or in ZG hyperplasia adjacent to the dominant aldosteronomas (26, 29, 32, 33); these findings suggest that nodule formation and excess aldosterone production are two dissociated events, implying a two-hit hypothesis for APA formation (16, 34). The first hit causing a unilateral aldosteronoma or a dominant nodule adjacent to ZG hyperplasia may result from a somatic mutation in one of the genes described above, at least in approximately 60% of cases. Possible causes of the second hit that results in dysregulation in cellular proliferation/apoptosis accelerating adenoma formation could be due either to activation of the Wnt/β-catenin pathway (35, 36), PKA pathway, or to gene mutations such as ARMC5 (37) (Figure 1). However, the pathophysiology of progression from normal adrenal to APA and the causes of diffuse bilateral hyperplasias, either as BAH or in mild form adjacent to the dominant aldosteronoma, are still unknown. Aldosterone-producing cell clusters (APCCs), which have increased expression of CYP11B2, are nests of cells below the adrenal capsule. They protrude into cortisol-producing cells that are usually negative for CYP11B2 expression. Nishimoto et al. found that APCCs are common in normal adrenals, and they harbor a different mutational spectrum compared to APA suggesting that APCCs could be a precursor for APA (38). In addition, several hormones activating variable levels of eutopic or ectopic aberrant receptors (18) (Figure 1) as well as autocrine and paracrine regulatory mechanisms (17) can increase aldosterone secretion in PA (either APA or BAH) independently from the suppressed RAS (see later section).

Adrenocorticotropic hormone can stimulate aldosterone secretion acutely and transiently under normal conditions, but to a lesser extent than angiotensin II and potassium. ACTH is a 39-amino-acid peptide, which results from the cleavage of its proopiomelanocortin (POMC) precursor by prohormone convertases PC1/3 and may be further cleaved by PC2 to generate α-melanocyte-stimulating hormone (α-MSH) (39, 40). It is mainly produced in the anterior pituitary corticotropes, but is also produced in brain, adrenal medulla, skin, and placenta (41–43). ACTH can induce aldosterone production at lower doses than the ones needed for cortisol and DHEA production (44). Furthermore, ACTH stimulates aldosterone production acutely and sometimes chronically.

The explanation for the increased role of ACTH in the regulation of aldosterone in PA may be secondary to the overexpression and function of MC2R in this condition. The expression of MC2R mRNA was shown to be upregulated in human adrenocortical neoplasms specifically in functional adenomas in contrast to non-functioning adenomas and carcinomas (89). More specifically, a few studies have demonstrated increased eutopic expression of MC2R assessed by RT-PCR or transcriptome studies in resected aldosteronomas as compared to cortisol-secreting adenomas, non-functional adenomas, and adrenocortical carcinomas (90–92). A particularly pertinent informative study included 15 adrenal tumors (14 APA and 1 BAH); MC2R mRNA levels were increased by a mean of 3.9-fold in those tissues compared to normal adrenal (18). However great variability existed in the level of expression in each tumor as 4 had lower levels than normal (0.3-fold to 0.7-fold), while those with increased expression varied between 1.4- and 20.6-fold compared to normal. The data are limited to mRNA expression without available measurements at the protein levels (no good specific MC2R antibody), but correlated well with the in vivo increased response to ACTH administration. There is almost no data on MC2R expression in BAH as those patients are usually not operated, but in the only case with BAH studied by this group MC2R was 20-fold increased. These data appear to be compatible with the findings that the majority of patients stimulated with ACTH during AVS will have concordant results before and after ACTH as the majority overexpress MC2R; however, the 28% of discordant results we found (87) may be explained by cases where MC2R are relatively decreased in the dominant adenoma but is present in adjacent hyperplasia. This hypothesis remains to be validated in prospective studies.

Adrenocorticotropin hormone is not the exclusive trigger of aldosterone secretion since several other hormones have a role in the pathophysiology of PA in addition to the ion channels mutations. Serotonin plays a significant role in aldosterone synthesis in normal physiological and in PA. The administration of serotonin 5-HT₄ agonists such as metoclopramide, cisapride, and tegaserod resulted in higher stimulation of aldosterone in PA as compared to the physiological moderate increase in normal individuals (18, 93, 94). Whereas non-specific inhibitors of 5-HT such as cyproheptadine and ketanserin produced only minor and transient inhibition of aldosterone secretion in aldosteronomas (95, 96), specific 5-HT₄R antagonists such as GR113808 were potent inhibitors of basal- and cisapride-induced aldosterone secretion (93). Chromaffin cells, endothelial cells, nerve terminals, and cells of the immune system are localized in the immediate vicinity of ZG cells and can secrete various factors to control aldosterone secretion (97). Local release of 5-HT by perivascular mast cells (MC) can activate 5-HT₄R expressed in ZG cells and consequently stimulate aldosterone production (98). A role of MC in tumorigenesis was proposed (99, 100). The density of MC was found to be increased in APA tissues compared with normal adrenals (101). As the 5-HT₄R have been found to be overexpressed in the majority of APA (but variable as MC2R) (17, 18, 102) and the ligand may be locally overexpressed also, a paracrine loop of regulation of aldosterone production appears to be present.

The compelling evidence supporting that various aberrant GPCR are frequently expressed in bilateral macronodular adrenal hyperplasia and Cushing’s syndrome (103) led many researchers to investigate the presence of aberrant GPCR in PA. Adrenal production of aldosterone in APA and BAH was found to be under the influence of aberrant GPCR and their ligands, as demonstrated by in vivo and in vitro studies (104, 105). The expression of ectopic receptors, which are usually not expressed at significant levels in normal ZG cells include those for glucose-dependent insulinotropic peptide (GIPR) (106), luteinizing hormone/human chorionic gonadotropin (LH–hCG R) (18, 106–112), β-adrenergic receptors (β-AR) (113), vasopressin (V1-AVPR) (18, 106, 114, 115), glucagon (glucagon receptor), TRH (TRH R) (18, 112, 116) and Endothelin-1 ET_A and ET_B receptors (117). Using a microarray approach in 10 aldosteronomas compared with five normal adrenals and 13 cortisol-secreting adenomas, the six GCPRs with highest increase in expression included LHCGR, 5-HT₄R, GnRHR, glutamate receptor metabotropic 3, endothelin receptor ET_B receptors, and MC2R (92). Table 1 summarizes the different types of aberrant eutopic or ectopic GPCR involved in aldosterone excess in PA. Co-expression of multiple aberrant GPCR was also reported; renin-independent stimulation of aldosterone secretion was observed in vivo following mixed meal, oral glucose, or administration of GIP, vasopressin, and tegaserod in a patient with unilateral source of PA (106). On the other hand, co-secretion of aldosterone and cortisol due to aberrant expression of GPCR was noted; in a patient with BMAH and β-AR-aberrant expression, isoproterenol stimulated both cortisol and aldosterone production (113).

Activating somatic CTNNB1 (β-catenin) mutations have now been identified in tumors of three women with APAs, two who presented during pregnancy and one after menopause (118). All three had heterozygous activating mutations of CTNNB1 and expressed aberrant LHCG and GNRH receptors at levels 100-fold higher than in other APAs. It was shown that the CTNNB1 mutation led to activation of the WNT pathway; it was suggested that could be the cause of dedifferentiation of gonadal progenitor cells present in the adrenal tissues with increased expression of gonadal receptors. It is thought that the high levels of endogenous human chorionic gonadotropin (hCG) during pregnancy and of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) after menopause led to the identification of APAs in these patients (118).

It is currently unclear whether these aberrant regulatory secretory mechanisms by ACTH and other hormones and the overexpression of their GPCR in PA are secondary to unknown proliferative mechanisms or are primary and at least partially responsible for the abnormal proliferation, the initiation of diffuse BAH. However, they clearly play a role in aldosterone secretion which is not autonomous.

Our understanding of the increased occurrence and complexity of molecular etiology and unique signature in each case of PA has progressed greatly in recent years. The increased role of ACTH, of the variable expression of MC2R, and of other aberrant GPCR in PA should receive further attention in the future. The development of effective antagonists to MC2R and other aberrant GPCR could eventually offer interesting alternatives in patients with bilateral sources of excess aldosterone in combination with better antagonists of the mineralocorticoid receptor.

NG, IB, and AL contributed to the conception and design of the manuscript as well as to drafting the review article and they all provided final approval of the version to be published.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.