acute and chronic regulation of aldosterone production

Molecular and Cellular Endocrinology 350 (2012) 151–162

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Molecular and Cellular Endocrinology

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Review

Acute and chronic regulation of aldosterone production

Namita G. Hattangady a,1, Lawrence O. Olala a,1, Wendy B. Bollag a,b, William E. Rainey a,⇑a Department of Physiology, Georgia Health Sciences University (Formerly The Medical College of Georgia), 1120 15th Street, Augusta, GA 30912, USAb Charlie Norwood VA Medical Center, One Freedom Way, Augusta, GA 30904, USA

a r t i c l e i n f o

Article history:Available online 4 August 2011

Keywords:Aldosterone synthaseAngiotensin IIPotassiumAdrenocorticotropin

0303-7207/$ - see front matter � 2011 Elsevier Irelandoi:10.1016/j.mce.2011.07.034

⇑ Corresponding author. Tel.: +1 706 721 7665; faxE-mail address: [email protected] (W.E.

1 These authors contributed equally to the manuscri

a b s t r a c t

Aldosterone is the major mineralocorticoid synthesized by the adrenal and plays an important role in theregulation of systemic blood pressure through the absorption of sodium and water. Aldosterone produc-tion is regulated tightly by selective expression of aldosterone synthase (CYP11B2) in the adrenal outer-most zone, the zona glomerulosa. Angiotensin II (Ang II), potassium (K+) and adrenocorticotropin (ACTH)are the main physiological agonists which regulate aldosterone secretion. Aldosterone production is reg-ulated within minutes of stimulation (acutely) through increased expression and phosphorylation of thesteroidogenic acute regulatory (StAR) protein and over hours to days (chronically) by increased expres-sion of the enzymes involved in the synthesis of aldosterone, particularly CYP11B2. Imbalance in any ofthese processes may lead to several disorders of aldosterone excess. In this review we attempt to sum-marize the key molecular events involved in the acute and chronic phases of aldosterone secretion.

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1512. Steroidogenesis (Aldosterone production) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513. Acute effects of AngII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1534. Acute effects of potassium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1565. Acute effects of ACTH. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1566. Chronic effects of AngII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1567. Chronic effects of potassium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1578. Chronic effects of ACTH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1589. Lessons from primary aldosteronism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15810. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

1. Introduction

Aldosterone is the major mineralocorticoid involved in main-taining fluid and electrolyte balance in all mammals. In humans,excessive secretion of this hormone results in hypertension, con-tributes to cardiac fibrosis, congestive heart failure, and exacer-bates the morbidity and mortality associated with thesedisorders (Gekle and Grossmann, 2009; Marney and Brown,

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2007). Although the signal transduction processes regulating aldo-sterone production are as yet incompletely understood, ongoingresearch has identified several important pathways mediating ste-roidogenesis. Aldosterone production (equivalent to secretion inthe case of this steroid hormone) is primarily regulated by angio-tensin II (AngII), serum potassium, as well as adrenocorticotropichormone (ACTH).

2. Steroidogenesis (Aldosterone production)

In mammals, aldosterone biosynthesis occurs almost solely inthe adrenal zona glomerulosa. Aldosterone is derived through a

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Fig. 1. Adrenocortical steroidogenic pathways for the production of mineralocorticoids and glucocorticoids. The adrenal cortex produces zone-specific steroids as a result ofdifferential expression of steroidogenic enzymes. In the initial step of steroidogenesis, steroidogenic acute regulatory (StAR) protein is needed for the rate-limiting stepof movement of cholesterol to the inner mitochondrial membrane, where cholesterol is cleaved by cholesterol side-chain cleavage (CYP11A1) to pregnenolone. Further stepsof the steroidogenic pathway include the enzymes 3b-hydroxysteroid dehydrogenase type 2 (HSD3B2), 17a-hydroxylase, 17,20-lyase (CYP17), 21-hydrolylase (CYP21), 11b-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2).

152 N.G. Hattangady et al. / Molecular and Cellular Endocrinology 350 (2012) 151–162

series of enzymatic steps that involve three cytochrome P450 en-zymes and one hydroxysteroid dehydrogenase (Fig. 1). The en-zymes cholesterol side-chain cleavage (CYP11A1), 21-hydroxylase(CYP21) and aldosterone synthase (CYP11B2) belong to the cyto-chrome P450 family of enzymes. CYP11A1 and CYP11B2 are local-ized to the inner mitochondrial membrane, while CYP21 is found inthe endoplasmic reticulum. Cytochrome P450 enzymes are heme-containing proteins that accept electrons from NADPH viaaccessory proteins and utilize molecular oxygen to performhydroxylations (CYP21 and CYP11B2) or other oxidative conver-sions (CYP11A1). The fourth enzyme, type 2 3b-hydroxysteroiddehydrogenase (HSD3B2), is a member of the short-chain dehydro-genase family and is localized in the endoplasmic reticulum. Aldo-sterone and cortisol share the first few enzymatic reactions in theirbiosynthetic pathways (cholesterol to progesterone); however,adrenal zone-specific expression of CYP11B2 (aldosterone syn-thase) in the glomerulosa and that of CYP11B1 (11b-hydoxylase)in the fasciculata leads to the functional zonation observed in theadrenal cortex (Rainey, 1999).

Like all steroid hormones, the glomerulosa cell uses cholesterolas the primary precursor for steroidogenesis. The cholesterolneeded for adrenal steroidogenesis can come from several sources,which include de novo synthesis from acetate or cholesteryl estersstored in lipid droplets or up-take by the low-density lipoprotein(LDL) receptor (for LDL) or scavenger receptor-BI (for high-densitylipoprotein or HDL). Movement of cholesterol from the outer mito-chondrial membrane, across the aqueous intra-membranous space,to the inner mitochondrial membrane must occur for CYP11A1 toaccess the molecule for cleavage to pregnenolone. Because steroidhormones are secreted upon synthesis, the initial reaction involv-ing mitochondrial conversion of cholesterol to pregnenolone istightly controlled and represents the rate-limiting step in aldoste-rone synthesis. This step is regulated by the expression and phos-phorylation of steroidogenic acute regulatory protein (StAR)(Arakane et al., 1997; Fleury et al., 2004; Manna et al., 2009). Preg-nenolone passively diffuses into the endoplasmic reticulum and is

converted to progesterone by HSD3B2. Progesterone is hydroxyl-ated to deoxycorticosterone by CYP21. Finally, aldosteronebiosynthesis is completed in the mitochondria, where deoxycorti-costerone undergoes 11b- and 18-hydroxylation, followed by18-oxidation, which in humans can be mediated by a single en-zyme, CYP11B2. Although the last step of cortisol production alsoinvolves the 11-hydroxylation of 11-deoxycortisol to cortisol by11b-hydoxylase, this enzyme only poorly catalyzes the 18-hydrox-ylation reaction and does not catalyze 18-oxidation.

There are several factors regulating aldosterone production inthe adrenal zona glomerulosa. First, the selective expression ofCYP11B2 in the glomerulosa creates a tightly controlled zone-specific ability to make aldosterone and limits production of thesteroid outside of this relatively small adrenal zone (Domaliket al., 1991; Ogishima et al., 1992; Pascoe et al., 1995). In ratsand mice CYP11B2 is expressed in a tight zonal pattern that circlesthe adrenal (Domalik et al., 1991; Ogishima et al., 1992). A recentstudy revealed a variation in human adrenal glomerulosa zonationcharacterized by the presence of relatively few subcapsular cellclusters expressing CYP11B2 (Nishimoto et al., 2010). This pheno-type may relate to the relatively high sodium diet and resultantsuppression of the renin-angiotensin system in most human popu-lations. It is hypothesized that these CYP11B2-expressing corticalcell clusters may be the precursors to aldosterone-producing ade-nomas (APA) (Nishimoto et al., 2010). The absence of CYP17 inglomerulosa cells is a second mechanism resulting in diversion ofthe steroidogenic pathway toward aldosterone (Narasaka et al.,2001). Finally, the centripetal blood flow in the adrenal cortex,itself, prevents the precursors of aldosterone in the fasciculatacells from accessing CYP11B2 in the zona glomerulosa, therebyhelping to maintain the functional specificity of the adrenocorticalzones.

The regulation of glomerulosa aldosterone biosynthesis is di-vided into two key events in the steroidogenic pathway (Clarket al., 1992; Muller, 1998). Acutely (minutes after a stimulus), aldo-sterone production is controlled by rapid signaling pathways that

Fig. 2. Acute actions of AngII, K+ and ACTH on adrenal glomerulosa cell aldosterone production. AngII binds the AT1 receptor to activate phosphoinositide-specificphospholipase C (PLC)-mediated cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 binds the IP3R onthe endoplasmic reticulum (ER), releasing calcium and raising cytosolic calcium concentrations. AngII also activates, in part through protein kinase C (PKC), phospholipase D(PLD), which hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA) which can be metabolized to DAG by lipid phosphate phosphatases. Small increases inextracellular K+ depolarize the glomerulosa cell, activating the voltage-operated L-and T-type calcium channels, increasing calcium influx. Increased intracellular calciumconcentration activates calcium/calmodulin-dependent protein kinases I/II (CaMK), as well as PKC isoforms. Both of these pathways can modulate not only StARphosphorylation, but also expression, likely in part through the StAR promoter binding of cAMP response element binding protein (CREB). The DAG/PKC pathway alsoactivates protein kinase D (PKD) which can likewise phosphorylate (and activate) CREB. DAG can be hydrolyzed by DAG lipase to release arachidonic acid, which can befurther metabolized by 12-lipoxygenase to 12-hydroxyeicosatetraenoic acid (12-HETE), which also induces the phosphorylation (and activation) of CREB. ACTH can alsomediate aldosterone synthesis through binding to the melanocortin type 2 receptor (MC2R), thus activating through a heterotrimeric Gs protein, adenylate cyclase (AC). ACconverts adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). cAMP activates protein kinase A (PKA) inducing a slow but sustained calcium influxthrough the L-type calcium channels. PKA also phosphorylates CREB, thereby increasing StAR expression. Cholesterol for aldosterone production arises from cholesteryl esterhydrolase (CEH)-mediated hydrolysis of cholesteryl esters synthesized de novo or obtained from lipoproteins and stored in lipid droplets. Free cholesterol is shuttled to theinner mitochondrial membrane by StAR likely in complex with other cholesterol transport proteins.

N.G. Hattangady et al. / Molecular and Cellular Endocrinology 350 (2012) 151–162 153

increase the movement of cholesterol into the mitochondria whereit is converted to pregnenolone. This has been called the ‘‘early reg-ulatory step’’ and is mediated by increased expression and phos-phorylation of StAR protein (Fig. 2) (Arakane et al., 1997;Cherradi et al., 1998; Fleury et al., 2004; Manna et al., 2009).Chronically (hours to days), aldosterone production is regulatedat the level of expression of the enzymes involved in the synthesisof aldosterone (Fig. 3) (Bassett et al., 2004b). This has been calledthe ‘‘late regulatory step’’ and is particularly dependent on in-creased expression of CYP11B2. The goal of this review article isto summarize our current understanding of the signaling pathwaysthat regulate the early and late regulatory steps of aldosteronebiosynthesis.

3. Acute effects of AngII

As mentioned above, the initial enzymatic step in aldosteronebiosynthesis is the hydrolysis of cholesterol to pregnenolone bythe cholesterol side-chain cleavage complex found in the innermitochondrial membrane. Thus, initiation of aldosterone produc-tion in response to agonists such as AngII, elevated extracellular

potassium concentrations or ACTH requires two major steps: first,cholesterol mobilization from lipid droplets to the mitochondria isthought to require cytoskeletal rearrangements (Crivello and Jefco-ate, 1979; Crivello and Jefcoate, 1980; Feuilloley and Vaudy, 1996)and activation of cholesteryl ester hydrolase by, e.g., extracellularsignal-regulated kinase-1 and -2 (ERK-1/2) (Cherradi et al., 2003).Mobilization is followed by movement of the cholesterol fromthe outer to the inner mitochondrial membrane, a process requir-ing active StAR protein (Clark et al., 1994; Krueger and Orme-John-son, 1983; Krueger and Orme-Johnson, 1988; Pon and Orme-Johnson, 1985; Pon and Orme-Johnson, 1986). As discussed below,StAR appears to be regulated both at the level of transcription (Joet al., 2005; Manna et al., 2009) and post-translationally, in thatphosphorylation appears necessary for its full activity(Arakaneet al., 1997; Fleury et al., 2004; Manna et al., 2009; Stocco et al.,2005).

Both of these processes are triggered, in the case of AngII, by thebinding of the hormone to the type 1 AngII (AT1) receptor and theactivation of several signaling pathways (Fig. 2). One such pathwayis a phosphoinositide-specific phospholipase C (PLC), which hydro-lyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate thetwo second messengers, inositol 1,4,5-trisphosphate (IP3) and

Fig. 3. The chronic production of aldosterone is regulated by AngII and potassium (K+). AngII binds type 1 AngII receptors (AT1-R) and activates phospholipase C (PLC) whichcauses hydrolysis of phosphatidylinositol-4,5-bisphosphate to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG activates protein kinase C (PKC) which inhibitstranscription of 17a-hydrolyase (CYP17) through transcription factors such as cFOS. DAG may also increase the activity of protein kinase D (PKD), which has been shown toincrease CYP11B2 transcription. IP3 causes the release of intracellular calcium and the activation of calcium-calmodulin kinases (CaMKs). Small increases in extracellular K+

also depolarize the glomerulosa cell, increasing calcium influx and activating CaMKs. CaMKs increase expression and/or phosphorylation and activation of transcriptionfactors that increase CYP11B2 transcription. Ang II also increases the expression of LDL and HDL receptors, which increases cholesterol availability for steroidogenesis.


diacylglycerol (DAG) (Barrett et al., 1989; Bird et al., 1993; Bollaget al., 1991; Farese et al., 1981; Ganguly and Davis, 1994; Hunyadyet al., 1990; Kojima et al., 1984a). IP3 is thought to initiate aldoste-rone secretion by eliciting a transient increase in the cytosolic cal-cium concentration and activating calcium/calmodulin-dependentprotein kinases (CaMK). CaMK activity is clearly important inmediating aldosterone secretion, as inhibition of this enzyme de-creases AngII-induced aldosterone secretion (Ganguly et al.,1992; Pezzi et al., 1996; Spät and Hunyady, 2004). On the otherhand, DAG functions to stimulate protein kinase C (PKC), the activ-ity of which has been suggested by some groups to underlie sus-tained aldosterone secretion from bovine glomerulosa cells(Bollag et al., 1990; Bollag et al., 1992; Bollag et al., 1991; Kapaset al., 1995; Kojima et al., 1984a). This idea is supported by theability of phorbol esters (Betancourt-Calle et al., 1999; Kojimaet al., 1985d), a DAG-generating enzyme (Bollag et al., 1990) andsynthetic DAGs (Kojima et al., 1985d) to elicit aldosterone secre-tion and of a selective PKC inhibitor to decrease AngII-inducedaldosterone secretion (Kapas et al., 1995). However, other groupshave suggested an inhibitory role of PKC in the aldosterone secre-tory response as discussed below (Aptel et al., 1996; Hajnoczkyet al., 1992; Rossier et al., 1995).

There are two additional pathways activated by AngII binding tothe AT1 receptor, through mechanisms that are as yet incompletelydefined. The first is an increase in calcium influx, in part throughCaM kinase II (Barrett et al., 2000; Fern et al., 1995; Lu et al.,1994; Yao et al., 2006) and GTP-binding proteins (Lu et al., 1996),with this enhancement acting synergistically with PKC-activatingagents to stimulate secretion. Indeed, this calcium influx is essen-tial for a continued aldosterone secretory response (Barrett et al.,

1989; Ganguly and Davis, 1994), as well as for regulating PKCactivity (Kojima et al., 1994), since inhibition of calcium influx withcalcium channel antagonists decreases AngII-induced aldosteronesecretion (Kojima et al., 1985c; Rossier et al., 1996; Rossier et al.,1998; Spät and Hunyady, 2004). Calcium influx occurs throughvoltage-dependent T- and L-type calcium channels, as well asstore-operated calcium channels or capacitative calcium influx(Aptel et al., 1999; Burnay et al., 1994; Burnay et al., 1998; Spätand Hunyady, 2004). The activity of the voltage-dependent chan-nels is maintained by appropriate membrane polarization, main-tained by proper functioning of potassium channels, such asTWIK-related acid-sensitive K (TASK) channels (Davies et al.,2008; Nogueira et al., 2010b). Emptying of the endoplasmic reticu-lum store by IP3 also results in capacitative calcium influx, as dis-cussed above. The importance of this calcium signal to acutealdosterone secretion, functioning largely through activation ofCaMK, is well appreciated and has been reviewed thoroughly(Spät and Hunyady, 2004).

On the other hand, the role of DAG effector enzymes such asPKC, in modulating aldosterone synthesis remains somewhat con-troversial, with some reports showing that activation of the PKCpathway enhances aldosterone secretion, and others that thispathway inhibits aldosterone production (Hajnoczky et al., 1992;Kojima et al., 1984a; Kojima et al., 1983; LeHoux et al., 2001;LeHoux and Lefebvre, 2006; Lehoux and Lefebvre, 2007). Thus,some investigators have shown that mimicking the PKC and cal-cium influx signals with pharmacologic agents essentially repro-duces AngII responses including aldosterone secretion (Bollaget al., 1990; Kojima et al., 1984a; Kojima et al., 1985c) and the pro-tein phosphorylation pattern (Barrett et al., 1986). These results


suggest that the aldosterone secretory response to AngII requiresboth a PKC activation event and a calcium influx stimulus. In con-trast, there is evidence to indicate that the DAG signal and its effec-tors oppose aldosterone secretion acutely in rat glomerulosa cells,with inhibition of PKC by pretreatment with the non-selectiveinhibitor staurosporine enhancing subsequent AngII-induced aldo-sterone secretion (Hajnoczky et al., 1992). This result has been con-firmed in experiments with the selective PKC inhibitorsbisindolymaleimide I and Gödecke 6976 (LeHoux et al., 2001).Other experiments have observed essentially no effect of DAG-mimicking phorbol esters on more chronic aldosterone secretionfrom the human adrenocortical carcinoma cell line, H295R (Birdet al., 1995b). One possible explanation arises from the lack ofselectivity/specificity of phorbol esters as mimics of the DAG path-way and/or PKC inhibitors as blockers of the pathway (see below).Clearly, additional studies are required to define the role of DAG-mediated signaling in AngII-induced aldosterone synthesis.

Another signaling system activated by AngII is phospholipase D(PLD), which can also increase DAG (indirectly) and presumablyactivate PKC (Bollag et al., 1990; Bollag et al., 2002; Zheng andBollag, 2003). PLD, of which there are two well-characterizedmammalian isoforms PLD1 and PLD2, hydrolyzes phospholipids,primarily phosphatidylcholine, to yield phosphatidic acid (phos-phorylated DAG), which can then be converted to DAG by the ac-tion of lipid phosphate phosphatases (Bollag et al., 2007; Qinet al., 2010). Phosphatidic acid is a second messenger itself and isproposed to function as a slow-release reservoir of diacylglycerolfor sustained cellular responses (Bollag et al., 1990; Bollag et al.,2007; Bollag and Xie, 2008). Phosphatidic acid can also be deacy-lated by phospholipase A2 to produce lysophosphatidic acid(LPA), a lipid message that activates G protein-coupled LPA recep-tors. The released fatty acid can also be metabolized to additionallipid signals, such as eicosanoids and 12-hydroxyeicosatetraenoicacid (12-HETE), which has been reported to stimulate aldosteronesecretion (Natarajan et al., 1990; Natarajan et al., 1988). Treatmentof glomerulosa cells with exogenous PLD alone or in combinationwith the calcium channel agonist, BAY K8644, induces a sustainedincrease in aldosterone secretion without an increase in phospho-inositide hydrolysis (Bollag et al., 1990), suggesting that PLD activ-ity may be sufficient to stimulate aldosterone secretion. PLDactivity also appears to be necessary for AngII-induced aldosteronesecretion, as demonstrated using the primary alcohol 1-butanol,which diverts production away from phosphatidic acid and DAG(Su et al., 2009) and instead forms phosphatidylbutanol (versusthe control tert-butanol, which does not affect PLD-generated lipidsignals). 1-Butanol was found to inhibit the AngII-induced increasein DAG and phosphatidic acid levels (Bollag et al., 2002), as well asAngII-elicited aldosterone secretion, in bovine adrenal glomerulosacells (Bollag et al., 2002) and in H295R cells (Zheng and Bollag,2003), whereas tert-butanol did not (Zheng and Bollag, 2003).More direct evidence arises from studies in which PLD was overex-pressed using adenovirus-mediated transduction. Overexpressionof wild-type PLD1 or PLD2, but not the lipase-inactive isoforms, in-creased PLD activity (Qin et al., 2010). However, only wild-typePLD2 enhanced AngII-stimulated aldosterone secretion (Qin et al.,2010), suggesting that PLD2 is the isoform mediating aldosteronesecretion in response to AngII, although the lipid signal(s) mediat-ing this effect is unclear.

Thus, as discussed above, DAG can be produced by both AngII-activated phospholipase C and PLD and, in turn, can activate DAGeffector enzymes, which include PKC isoenzymes and protein ki-nase D (PKD), as well as Ras guanine nucleotide exchange factors(Ras-GRP1 through 3), which are upstream of the mitogen-acti-vated protein kinase pathway involving Ras, Raf, MEK and ERK-1/2, and chimaerins (GTPase-activating proteins for Rac) (Brose andRosenmund, 2002). PKC isoenzymes constitute a multigene family

that belongs to the cAMP-dependent protein kinase A/protein ki-nase G/protein kinase C (AGC) family (Mellor and Parker, 1998).Members of the family play an important role in mediating numer-ous intracellular signaling events, including those involved in cellgrowth and differentiation, membrane trafficking, secretion, andgene expression (Mellor and Parker, 1998; Spät and Hunyady,2004). The PKC family is divided into three groups based on theirsecond messenger requirements: the conventional (also knownas classical), novel, and atypical, with all members requiring acidicphospholipids such as phosphatidylserine for activation.Conventional and novel PKCs also require DAG and in addition con-ventional PKCs require calcium. Alpha, betaI, betaII and gammacomprise the conventional PKC subfamily, whereas novel PKCs in-clude the delta, epsilon, eta, and theta isoforms. The novel isoformshave been shown to activate protein kinase D (PKD) via phosphor-ylating serines in the catalytic domain (Waldron and Rozengurt,2003) (see below).

PKCs are also involved in regulating adrenal steroidogenesisthrough the phosphorylation of hydroxyl groups of serine andthreonine amino acid residues on substrate proteins (Betancourt-Calle et al., 1999; Kapas et al., 1995; Lang and Valloton, 1987;Nakano et al., 1990; Natarajan et al., 1994). There is evidence tosuggest that DAG activation of PKC coincides with the expressionof the rate-limiting StAR protein (Manna et al., 2009) as well asStAR phosphorylation (Betancourt-Calle et al., 2001a). In turn,phosphorylation of StAR has been proposed to be required for itsfull activity (Arakane et al., 1997; Fleury et al., 2004; Mannaet al., 2009). For example, using a mouse Leydig cell line, Stoccoand colleagues (Manna et al., 2009) showed that activation of thePKC pathway can increase StAR expression but has a minimal ef-fect on steroidogenesis, unless small concentrations of dibutyryl-cAMP are also included. Thus, these low doses of cAMP act syner-gistically with a PKC-activating phorbol ester to stimulate StARphosphorylation and maximal steroid hormone production. Bollagand colleagues have also demonstrated that treatment of H295Rcells with the DAG-mimicking phorbol ester, phorbol 12-myristate13-acetate (PMA) increases mitochondrial cholesterol levels andaldosterone secretion (Bollag et al., 2008), providing evidence fora possible role of PKC (and/or other DAG effector enzymes) in cho-lesterol transport and aldosterone production.

The 18 kDa mitochondrial protein peripheral type benzodiaze-pine receptor (PBR), also known as translocator protein (TSPO),has also been implicated in steroidogenesis (Jefcoate, 2002;Papadopoulos et al., 2007). PBR has been proposed to functionallyinteract with StAR, among other proteins, to mediate mitochon-drial cholesterol uptake into the mitochondria (Hauet et al.,2002). PBR is located on the outer mitochondrial membrane andis elevated in steroidogenic cells upon hormone stimulation (Hauetet al., 2002). Introduction of a null mutation of the PBR gene intoR2C rat Leydig tumor cells inhibited pregnenolone production inresponse to 22R-hydroxycholesterol, a precursor that bypassesthe need for StAR (Papadopoulos et al., 2007), supporting an impor-tant role for this protein in steroidogenesis. Recently, the DAG-sensitive PKC isozyme, PKC-epsilon was shown to mediate PBRgene expression (Batarseh et al., 2008) in MA-10 Leydig cells usingPKC inhibitors and small interfering (si)RNA targeting PKC-epsilon.These results may provide insight into the possible role of the PKCpathway in mediating aldosterone secretion, although additionalstudies are certainly required to fully define the role(s) of this largefamily of serine/threonine protein kinases.

The PKD family of enzymes, composed of PKD1 (also known asPKC-l), PKD2 and PKD3 (also known as PKC-m), represent DAG-activated serine/threonine kinases downstream of novel PKCsand a Src family tyrosine kinase pathway. An involvement of PKDfamily members has been proposed for a number of important cel-lular functions, including Golgi trafficking, hypertrophy, immune


response, proliferation, migration, invasion and survival. (Guhaet al., 2010; Lavalle et al., 2010), Importantly, PKD is activated inresponse to AngII in primary bovine adrenal glomerulosa cells(Shapiro and Bollag, 2004). Moreover, adenovirus-mediated over-expression of a constitutively active PKD1 mutant enhances, anda dominant-negative PKD1 mutant inhibits acute AngII-inducedaldosterone secretion in these cells (Shapiro and Bollag, 2004).PKD performs a similar role to enhance chronic AngII-inducedaldosterone secretion and aldosterone synthase expression in theH295R cell model (Romero et al., 2006), a result which was corrob-orated by a study showing that knocking down PKD levels withRNA interference decreased AngII-stimulated aldosterone secre-tion from these cells (Chang et al., 2007). Thus, PKD appears tomediate AngII-elicited aldosterone secretion acutely as well as toregulate aldosterone biosynthetic capacity of the adrenal. Also inthe H295R cells, PKD activation in response to AngII resulted fromPKC-epsilon-mediated transphosphorylation (Romero et al., 2006).While the mechanism by which PKD enhances aldosterone secre-tion is unclear, it is known that StAR gene transcription is influ-enced by several transcription factors that are targets for PKD,including cAMP response-element (CRE)-binding protein (CREB)/CRE modulator (CREM)/activating transcription factor (ATF) familymembers and activator protein-1 (AP-1, Fos/Jun) (Bassett et al.,2004a; Gonzalez and Montminy, 1989; Manna et al., 2003; Stoccoet al., 2005). Indeed, Stocco and colleagues recently showed thatPKD regulates StAR levels via CREB and AP-1 in MA-10 Leydig cells(Manna et al., 2011).

4. Acute effects of potassium

Similar to AngII, small increases in extracellular potassium lev-els also stimulate calcium influx, via depolarization of the glome-rulosa cell plasma membrane and activation of the voltage-dependent calcium channels, transient T-type and long-lasting L-type. Also as with AngII, this influx is required for the response topotassium, since inhibition of calcium influx abolishes potassium-stimulated aldosterone secretion (Capponi et al., 1984; Kojimaet al., 1985a; Kojima et al., 1984b; Rossier et al., 1998; Spät andHunyady, 2004). Related to calcium influx, an interesting findingis the fact that decreasing potassium levels to 2 mM inhibits An-gII-stimulated aldosterone production, presumably by inhibitingAngII-induced calcium influx (Kojima et al., 1985d). It can be spec-ulated that this mechanism serves under conditions of low serumpotassium levels to prevent AngII-stimulated aldosterone secre-tion, which could otherwise cause excessive excretion of potassiumand subsequent severe, and possibly fatal, hypokalemia.

Other signaling pathways have also been proposed to be involvedin potassium-induced aldosterone production, although contro-versy remains concerning these other events. For instance, someinvestigators have observed an ability of potassium to increasecAMP levels and others have not (Fujita et al., 1979; Ganguly et al.,1990; Kojima et al., 1985b; Tait and Tait, 1999). Potassium has alsobeen reported to stimulate PLD activity through voltage-dependentcalcium channels in bovine glomerulosa cells (Betancourt-Calleet al., 2001b), although whether and how this PLD activation contrib-utes to potassium-elicited aldosterone production is unclear. Thus,there are still several areas related to the acute effects of potassiumon aldosterone secretion in adrenal glomerulosa cells that still needto be studied and better understood.

5. Acute effects of ACTH

ACTH is able to stimulate aldosterone production acutely, bothin vivo and in vitro. It does so by binding to the ACTH receptor(MC2R) which activates adenylate cyclase via the heterotrimeric

G protein, Gs. Adenylate cyclase produces the second messengercAMP, thereby stimulating the activity of cAMP-dependent proteinkinase or protein kinase A (PKA). PKA can then phosphorylate andactivate StAR (Betancourt-Calle et al., 2001a), to increase choles-terol delivery to the inner mitochondrial membrane. In addition,PKA can regulate the transcriptional activity of CREB family tran-scription factors (Jo et al., 2005; Johannessen and Moens, 2007a;Manna et al., 2011). Since binding sequences for CREB/ATF tran-scription factors are found in the promoter of StAR (Bassett et al.,2000; Clem et al., 2005; Manna et al., 2002; Manna et al., 2003;Sher et al., 2007), PKA can also rapidly increase expression of StARand further enhance acute steroid production. In addition to stim-ulating cAMP-induced PKA activation, ACTH is capable of promot-ing calcium influx, likely through PKA-mediated phosphorylationof L-type calcium channels (Sculptoreanu et al., 1993), therebyincreasing cytosolic calcium concentration and further enhancingadenylate cyclase production of cAMP and aldosterone secretion(Gallo-Payet et al., 1996).

Finally, there may be some PKA-independent effects of cAMP onaldosterone secretion, in that, a recent report has suggested theinvolvement of the guanine nucleotide exchange factor, exchangeprotein directly activated by cAMP (Epac), in cAMP-mediated aldo-sterone production, via effects on CaMK activation (Gambaryanet al., 2006).

6. Chronic effects of AngII

As noted earlier, the capacity for aldosterone production is alsoregulated through the chronic action of the same factors thatacutely stimulate its biosynthesis. The chronic actions involvechanges in the size of the zona glomerulosa as well as glomerulosacell capacity to produce aldosterone. This review focuses on thesecond issue (glomerulosa cell differentiation) because of recentadvances in our understanding of the mechanisms regulating ste-roidogenic enzymes.

AngII increases the expression of enzymes required for aldoste-rone synthesis, particularly CYP11B2. In vivo models have beencrucial in describing such AngII effects. Sodium restriction experi-ments in rats indicate that activation of the renin-AngII system(most often by low sodium diets) induces the expression ofCYP11B2 in glomerulosa cells without affecting that of CYP11B1(Adler et al., 1993; Holland and Carr, 1993; Tremblay et al.,1992). This result confirms the capacity of AngII to specifically in-crease the production of aldosterone but not that of glucocorticoids(Holland and Carr, 1993; Tremblay et al., 1992). One explanationfor the zone-specific effects of AngII is the greater expression ofAT1 receptors in glomerulosa versus fasciculata cells (Breaultet al., 1996), and the inhibition of CYP11B2 expression by angioten-sin inhibitors or AT1 receptor blockers has been demonstrated(Kakiki et al., 1997). AngII can also increase CYP11B2 expressionby increasing glomerulosa cell sensitivity to AngII through upregu-lation of its own receptor. This effect has been demonstratedin vivo in rats on a low-sodium diet (Du et al., 1996; Wang andDu, 1995); further studies have shown that pharmacological block-ers of the AT1 receptors, such as losartan (Du et al., 1996; Wangand Du, 1995) and angiotensin-converting enzyme (ACE) inhibitors(Lehoux et al., 1994; Wakamiya et al., 1994), reduce the effect ofthe RAS on the adrenal.

In vitro cell models have been particularly useful in defining theintracellular signaling mechanisms that lead to the chronic effectsof AngII. As with acute secretion, the best characterized pathwayregulating AngII-induced chronic production of aldosterone is thePLC-mediated generation of DAG and IP3, which increases intracel-lular calcium and acts via CaMK. Calcium signaling appears to bethe primary regulator of CYP11B2 transcription. However, since


inhibitors of calmodulin and CaMK cannot completely block AngII’sstimulation of CYP11B2 (Pezzi et al., 1997), other signaling mech-anisms are also likely involved. DAG-activated PKC, on the otherhand, does not appear to increase CYP11B2 transcript levels, butdoes play a role in producing an aldosterone-secreting glomerulosacell phenotype by inhibiting the expression of CYP17 (Bird et al.,1996; McEwan et al., 1999). This effect has been recently attrib-uted to Fos-mediated repression of the CYP17 transcriptional acti-vator, steroidogenic factor 1 (SF1) (Sirianni et al., 2010). On theother hand, there is evidence suggesting that certain PKC isoforms,such as PKC-epsilon may actually inhibit CYP11B2 expression viaactivation of ERK-1/2 (LeHoux and Lefebvre, 2006; Lehoux andLefebvre, 2007). However, PKC-epsilon has been shown to activatePKD, a kinase that can increase CYP11B2 expression (Romero et al.,2006) (see below), indicating that the role of the PKC family in reg-ulating chronic aldosterone production is as yet unclear.

AngII appears to increase CYP11B2 expression through the acti-vation of its transcription. A number of studies involving promoterdeletion and mutation analyses have revealed that cis-elements inthe CYP11B2 promoter are essential for basal as well as AngII-mediated CYP11B2 promoter activity. These include three key reg-ulatory cis-elements: one cAMP response element (CRE)/Ad1 andtwo distal cis-elements (Ad5 and NBRE) that are able to bind mem-bers of the nerve growth factor-induced clone B family of tran-scription factors (NGFI-B or NR4A family) (Bassett et al., 2004a;Bassett et al., 2004b; Bassett et al., 2000; Clyne et al., 1997;Nogueira and Rainey, 2010a; Romero et al., 2010; Szekeres et al.,2009). The over-expression of one of these transcription factors,NURR1 (NR4A2), has in fact been implicated in the developmentof aldosterone-producing tumors (Lu et al., 2004). Using multipleglomerulosa cell models, microarray studies have defined addi-tional transcription factors that are regulated by AngII (Nogueiraet al., 2009; Romero et al., 2007; Szekeres et al., 2010). The roleof these newly defined factors requires further study. Conversely,other transcriptional regulators such as SF1 (also called AD4BPand NR5A1) have been shown to repress basal and AngII-stimu-lated CYP11B2 expression (Bassett et al., 2002; Ye et al., 2009).However, it appears that the relative level of SF1 may decide itsability to repress CYP11B2 expression, since complete knock downof SF1 impairs the entire steroidogenic synthetic pathway (Ye et al.,2009). The underlying mechanism, however, is yet to be eluci-dated. As mentioned previously, yet another AngII-mediatedmechanism regulating chronic aldosterone secretion seems to bevia PKD (Romero et al., 2006). The pathway involved is undeter-mined in adrenal cells, although PKD activation of the transcriptionfactor CREB has been demonstrated in kidney HEK 293 cells(Johannessen et al., 2007b) and in MA-10 Leydig cells (Mannaet al., 2011). In the Leydig cell model PKD was shown to increaseStAR levels via effects on CREB and AP-1 transcription factors(Manna et al., 2011). At the level of cholesterol uptake in glomerul-osa cells, AngII has been shown to up-regulate the expression ofLDL and HDL receptors (Pilon et al., 2003) and of enzymes involvedin cholesterol synthesis (Liang et al., 2007).

Finally, AngII also increases aldosterone production by expan-sion of the zona glomerulosa via hypertrophy and hyperplasia. Thiseffect has been demonstrated in vivo, in rats on low-sodium diets,which display glomerulosa cell hypertrophy and proliferation inAT1R-dependent and-independent processes (McEwan et al.,1999). In vitro, primary cultures of bovine glomerulosa cells alsoshowed similar responses to AngII (Tian et al., 1995; Tian et al.,1998). These effects may be attributed to the ability of AngII to in-duce the expression of cyclin D1 (Watanabe et al., 1996). Mitogenicand hypertrophic effects of AngII have also been demonstrated inepithelial cells, vascular smooth muscle cells (Berk et al., 1989;Geisterfer et al., 1988), fibroblasts, cardiac myocytes (Sadoshimaand Izumo, 1993) and rat intestinal cells (Smith et al., 1994). It

can thus be speculated that the predisposition to cardiovasculardamage resulting from activation of the renin-angiotensin-aldoste-rone system can be attributed to AngII stimulation of not onlyaldosterone production pathways but also glomerulosa cell hyper-plasia and pathological growth of cardiovascular cells. Worth not-ing is the fact that in mice with targeted deletion of the renin/AngIIsystem, potassium can substitute for the effects of AngII to increaseadrenal expression of CYP11B2 and synthesis of aldosterone (Chenet al., 1997; Okubo et al., 1997). This result suggests that the mech-anisms of potassium and AngII stimulation of CYP11B2 expressionlikely overlap.

7. Chronic effects of potassium

Besides inducing early events to increase aldosterone produc-tion, potassium also regulates later/chronic events. It has been welldocumented that high potassium diets in rats increase the expres-sion of aldosterone synthase (CYP11B2) and aldosterone produc-tion (Tremblay and LeHoux, 1993; Tremblay et al., 1991). Arecent study in mice also reported a slight increase in the thicknessof the zona glomerulosa and suggested the role of several genes inthis process. These genes, including Mtus 1, Smoc 1 and Grp 48,were observed to be upregulated with 28 days of a high potassiumdiet, although the in vitro experiments did not completely parallelthese microarray results (Dierks et al., 2010). However, attributingthese observed changes solely to the chronic effects of potassiumon aldosterone production is particularly challenging, since physi-ological potassium levels are tightly regulated by the renin-angio-tensin system. Indeed, serum potassium levels following highpotassium diets were not reported to be abnormally high. Otherin vitro studies using primary cultures of rat glomerulosa cellsand the human adrenocortical H295R cell line have demonstratedincreased CYP11B2 mRNA levels, promoter activation and aldoste-rone biosynthesis in response to elevated potassium levels in thegrowth media (Bird et al., 1995b; Denner et al., 1996; Yagci andMuller, 1996b; Yagci et al., 1996a). These results may help to ex-plain the finding that in transgenic mice with targeted deletionin the renin-angiotensin system, potassium can induce CYP11B2expression in the adrenal as well as the synthesis of aldosterone(Chen et al., 1997; Okubo et al., 1997).

As mentioned earlier, the mechanism of potassium signaling inglomerulosa cells involves depolarization of the cells to allowextracellular calcium influx through the T- and L-type calciumchannels. This increase in calcium influx upregulates CYP11B2expression. Consistent with these findings, increasing calcium in-flux with the pharmacological calcium channel agonist BAYK8644increases CYP11B2 mRNA expression in the H295R cell model(Denner et al., 1996; Clyne et al., 1997; Pezzi et al., 1997). Further,this potassium-induced calcium influx elevation is abrogated bythe calcium channel blocker nifidipine (Denner et al., 1996; Yagciand Muller, 1996b), which also inhibits AngII-induced CYP11B2upregulation. As in the case of AngII, potassium-induced calciumsignaling occurs through the binding of calcium to the protein cal-modulin. The calcium-calmodulin (CaM) complex activates severalenzymes and kinases, many of which are expressed in a tissue-spe-cific manner. Amongst the different CaM kinases (CaMK), types Iand IV are more likely to be involved in chronic stimulation ofaldosterone secretion by AngII and potassium (Condon et al.,2002). Immunohistochemistry was used to demonstrate that CaM-KI expression is elevated in the glomerulosa of the human adrenalgland (Condon et al., 2002). The CaMK antagonist KN93 and thecalmodulin inhibitor calmidazolium effectively inhibit potas-sium-induced CYP11B2 mRNA upregulation (Bassett et al., 2004a;Pezzi et al., 1997) and promoter activation (Bassett et al., 2004b;Condon et al., 2002).


Another similarity that potassium shares with AngII is the abil-ity to activate several transcription factors, such as of NURR1, ATF1,ATF2 and CREB, which bind the proximal promoter of CYP11B2 atkey cis-elements to enhance transcription. Activation of these tran-scription factors appears to be mediated through phosphorylationby potassium-activated CaMK (Bassett et al., 2004a; Bassett et al.,2004b; Bassett et al., 2000; Nogueira and Rainey, 2010a). Support-ing these data is a recent study in which knock down of these tran-scription factors by siRNA technology reduced potassium-inducedCYP11B2 promoter activity and mRNA levels (Nogueira and Rainey,2010a). This idea is also supported by the observation that withinthe adrenal cortex, the transcription factor NURR1 has higherexpression in the zona glomerulosa and in aldosterone-producingtumors compared to the adjacent zona fasciculata (Lu et al., 2004).

As mentioned earlier, a small but interesting effect of a high-potassium diet in mice is the observed slight increase in the thick-ness of the zona glomerulosa (Gao et al., 2009). A more significantincrease in the thickness of the zona glomerulosa was found in ratson a high potassium diet for 2–7 weeks (Hartroft and Sowa, 1964).Also observed in the 2009 study by Gao et al. was elevated expres-sion of Gpr48, a G protein-coupled receptor that has been impli-cated in increasing tumor invasiveness and metastasis in theHeLa cervical carcinoma cell line (Gao et al., 2006; Gao et al.,2009). Moreover, Gpr48 is also associated with down-regulationof cyclin-dependent kinase inhibitor p27 (Kip1) (Gao et al.,2006). It is hence tempting to ascribe the observed increasedglomerulosa thickness to the effects of Gpr48. Chronic high potas-sium can thus regulate long-term aldosterone production, sodiumretention and ultimately blood pressure via chronic mechanismsinvolving increased glomerulosa cell size and/or number as wellas the cells, aldosterone synthetic capacity. However, a betterunderstanding of chronic effects of potassium on the molecularpathways underlying secretion of aldosterone by adrenal glome-rulosa cells is required.

8. Chronic effects of ACTH

While thought of primarily as the regulator of adrenal cortisolproduction, ACTH is considered a secondary regulator of zonaglomerulosa aldosterone production. It is clear that adrenal glome-rulosa cells (both in vivo and in vitro) can acutely increase aldoste-rone production in response to ACTH. However, over time ACTHcauses cultured glomerulosa cells to switch their phenotype to thatof a cortisol-producing fasciculata cell (Crivello and Gill, 1983;Hornsby et al., 1974). In vivo studies by Allen et al. demonstratedthat ablation of the pituitary pre-proopiomelanocortin-secretingcells that produce ACTH, and the resultant low ACTH level, wasaccompanied by a steep decrease in the transcript levels ofCYP11B1, but not of CYP11B2 (Allen et al., 1995). In agreementwith this observation, treatment with ACTH causes an initial in-crease in mRNA levels of CYP11B2 in the first 3 h; however, chron-ically, CYP11B2 expression decreases in response to ACTH in vitroin isolated rat adrenal cells (Holland and Carr, 1993). Similarly,chronic low-dose infusion of ACTH in human subjects results inan initial increase in plasma renin activity and plasma aldosteronelevels during the first 12–36 h, but a slow decline in these valuesover the next several days (Fuchs-Hammoser et al., 1980). Whilethe H295R adrenocortical cell lines express low levels of ACTHreceptors, treatment of these cells with cAMP analogs preferen-tially increases the expression of CYP11B1 over that of CYP11B2(Denner et al., 1996). The mechanism for chronic ACTH-mediatedrepression of aldosterone production and CYP11B2 expression re-mains unknown. An interesting observation has been that cAMPsignaling reduces the sensitivity of adrenocortical cells to AngIIby down-regulating the expression of AngII receptors (Bird et al.,

1995a; Yoshida et al., 1991). Another possible mechanism for thereduction in aldosterone production with chronic ACTH stimula-tion could be via the hormone’s direct induction of CYP11B1 andCYP17, the activities of which direct the precursors of the steroido-genic pathway away from the production of aldosterone, and to-wards that of cortisol (Bird et al., 1996).

Since CYP11B2 has a cAMP-regulatory element (CRE) in its 50

promoter region (Rainey, 1999), the mechanism preventing glome-rulosa cells from responding to ACTH with increased CYP11B2 andexcessive aldosterone production is not clear, but two possiblemechanisms have been suggested thus far. First, at least in bovineglomerulosa cells, there is high expression of the inhibitory guan-ine nucleotide-binding protein Gi. AngII signaling through the AT1

receptor couples through Gi to inhibit ACTH-stimulated cAMP for-mation (Begeot et al., 1988; Hausdorff et al., 1987). Second, adrenalglomerulosa cells appear to express adenylyl cyclases 5 and 6, iso-forms which are inhibited by a rise in intracellular calcium, a sig-naling mechanism common to AngII and potassium stimulationof aldosterone secretion (Shen et al., 2001) The above provide evi-dence for a supportive, but not an obligatory, role of ACTH in aldo-sterone production.

9. Lessons from primary aldosteronism

Primary aldosteronism (PA) is the major endocrine cause ofhypertension and is characterized by normal or elevated aldoste-rone levels in the presence of low circulating plasma renin levels(Young, 2007). The most common cause of PA is idiopathic hyper-aldosteronism (IHA), which occurs in approximately 60% of PA pa-tients. The second most abundant cause of PA is unilateraladrenocortical adenoma, which is a curable form of PA that canbe resolved by adrenalectomy. There are also rare forms of PA thatinclude glucocorticoid-remediable aldosteronism (GRA), or familialhyperaldosteronism 1 (FH1), which is caused by unequal crossoverbetween the CYP11B1 and CYP11B2 genes resulting in a hybridgene with the CYP11B1 promoter driving CYP11B2. In these casesCYP11B2 expression and aldosterone secretion are regulated byACTH in the adrenal zona fasciculata. Recently, several mousemodels have been developed in an attempt to study PA.

Two recent studies using different mouse models, both with adeletion of genes encoding TWIK-related acid-sensitive K (TASK)channels, have provided interestingly different and complex pri-mary aldosteronism phenotypes (Davies et al., 2008; Heitzmannet al., 2008). As mentioned earlier, TASK channels maintain themembrane potential of the glomerulosa cell at a polarized�70 mV by being constitutively open and acting as a K+ leak chan-nel. Inhibition of these channels by the AT1R or by increased serumK+ levels depolarizes glomerulosa cells and increases calcium in-flux to drive aldosterone secretion. In the study by Heitzmannet al., deletion of the TASK-1 channel resulted in a phenotypesimilar to the pathology of GRA, with characteristics such as salt-insensitive hyperaldosteronism, hypokalemia and dexametha-sone-suppressible aldosterone secretion. The deletion of TASK-1also seemed to change adrenal zonation and expression ofCYP11B2, which was absent in the outermost zona glomerulosabut was expressed to a large extent in the zona fasciculata. Fur-thermore, this expression pattern seemed to be restricted only tofemales and to males prior to puberty. On the other hand, Daviesand colleagues found that deletion of both TASK-1 and TASK-3 cre-ated a model with a phenotype resembling the pathology of IHA.Male mice showed increased aldosterone secretion that was notsuppressible with a high-salt diet or the AT1R blocker candesartan(Davies et al., 2008). Further studies will be required to understandthe mechanism by which the different types of TASK channelsinteract and regulate adrenal function.


Besides TASK channels, Choi et al. have also identified bothgerm-line and somatic mutations that occur near the selectivity fil-ter of the inward rectifying potassium channel KCNJ5 (Kir3.4) to re-sult in PA (Choi et al., 2011). The amino acid substitutions resultingfrom these mutations modified channel ion selectivity, such thatthe channel became permeable to both sodium and potassium,which led to increased depolarization of adrenocortical cells. Thisdepolarization is believed to cause elevated intracellular calciumand thereby the production of aldosterone and cell proliferation(Choi et al., 2011). These findings are particularly relevant in thatalmost 40% of aldosterone-producing adenomas appeared to havesuch a mutation in KCNJ5. Additional studies will be needed todetermine the exact mechanisms through which these mutationscause expansion of aldosterone-producing cells and formation ofadenomas.

10. Summary

Aldosterone is an essential hormone with key roles in the regu-lation of electrolyte balance and blood pressure. Its normal physi-ological regulators include Ang II, K+ and ACTH which can increasealdosterone secretion both acutely, by increasing StAR expressionand phosphorylation, as well as chronically, by action on the ste-roidogenic pathway, mostly through increasing gene expressionof CYP11B2. Dysregulation in aldosterone secretion, as is seen inprimary aldosteronism, leads to pathologies such as hypertensionand cardiovascular disease. Several animal and cell culture modelsare being developed to better understand aldosterone secretionunder normal and pathological conditions. The recent develop-ment of unique mouse models of primary aldosteronism and thediscovery of the KCNJ5 mutations as a cause of human PA havebeen particularly helpful in providing new clues to the mecha-nisms controlling aldosterone production and the adrenal glome-rulosa cell phenotype. However, additional studies will beneeded to define completely the detailed pathways that activateand repress aldosterone biosynthesis.

Acknowledgements

The authors were supported by National Institutes of Healthaward HL70046 (WBB) and a VA Merit Award (WBB). This workwas also supported by National Institutes of Health Grants DK-43140 and DK-069950 (to WER), a Synergy Award from the Cardio-vascular Discovery Institute, Georgia Health Sciences University (toWER).

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acute and chronic regulation of aldosterone production

Documents

aldosterone biosynthesis

synthesis of aldosterone

aldosterone production

acute effects of angii

acute effects of potassium5

acute effects of acth6

chronic effects of angii7

chronic effects of potassium8