Figure 1. Schematic of the adrenal gland. The adrenal gland is composed of the outer adrenal cortex and the inner adrenal medulla. The adrenal cortex is, in turn, composed of three layers: the innermost layer, the zona reticularis synthesizes and secretes adrenal androgens, such as dehydroepiandrosterone (DHEA) and DHEA‐sulfate. The middle layer of the adrenal cortex, the zona fasciculata, synthesizes, and secretes the glucocorticoid cortisol. (In rodents the glucocorticoid produced by the zona fasciculata is corticosterone.) The outer layer subjacent to the fibrous capsule delimiting the adrenal gland is the zona glomerulosa, which secretes the mineralocorticoid aldosterone. The adrenal medulla secretes the catecholamines epinephrine and norepinephrine.

Figure 2. Aldosterone biosynthesis. (A) In this schematic is shown the enzymatic process through which aldosterone is synthesized from its cholesterol precursor. The steroidogenic process is initiated following the transport of free cholesterol, usually released by cholesterol ester hydrolase (CEH), also known as hormone‐sensitive lipase, from cholesteryl esters stored in lipid droplets but also arising from de novo synthesis and uptake from circulating lipoproteins, to the outer mitochondrial membrane. From there the cholesterol is translocated to the inner mitochondrial membrane under the control of the steroidogenic acute regulatory (StAR) protein, which is the early rate‐limiting step in steroidogenesis, with the participation of other proteins (see Table 1). CYP11A1, also known as the cholesterol side‐chain cleavage complex, located in the inner mitochondrial membrane (IMM) initiates steroidogenesis by cleaving the side chain of cholesterol to produce pregnenolone. Pregnenolone is then metabolized by 3β‐hydroxysteroid dehydrogenase (3βHSD) in the endoplasmic reticulum (ER) to progesterone, which is, in turn, converted to 11‐deoxycorticosterone by 21‐hydroxylase (CYP21), also in the ER. Aldosterone synthase, or CYP11B2, then completes the synthesis of aldosterone in the mitochondria by catalyzing an 11β‐hydroxylation reaction, followed by the hydroxylation of carbon 18 and subsequent oxidation of the carbon 18‐hydroxy group to an aldehyde, to yield aldosterone. (B) Aldosterone biosynthesis is shown with the structure of each steroid illustrated.

Figure 3. Aldosterone biosynthesis. (A) In this schematic is shown the enzymatic process through which aldosterone is synthesized from its cholesterol precursor. The steroidogenic process is initiated following the transport of free cholesterol, usually released by cholesterol ester hydrolase (CEH), also known as hormone‐sensitive lipase, from cholesteryl esters stored in lipid droplets but also arising from de novo synthesis and uptake from circulating lipoproteins, to the outer mitochondrial membrane. From there the cholesterol is translocated to the inner mitochondrial membrane under the control of the steroidogenic acute regulatory (StAR) protein, which is the early rate‐limiting step in steroidogenesis, with the participation of other proteins (see Table 1). CYP11A1, also known as the cholesterol side‐chain cleavage complex, located in the inner mitochondrial membrane (IMM) initiates steroidogenesis by cleaving the side chain of cholesterol to produce pregnenolone. Pregnenolone is then metabolized by 3β‐hydroxysteroid dehydrogenase (3βHSD) in the endoplasmic reticulum (ER) to progesterone, which is, in turn, converted to 11‐deoxycorticosterone by 21‐hydroxylase (CYP21), also in the ER. Aldosterone synthase, or CYP11B2, then completes the synthesis of aldosterone in the mitochondria by catalyzing an 11β‐hydroxylation reaction, followed by the hydroxylation of carbon 18 and subsequent oxidation of the carbon 18‐hydroxy group to an aldehyde, to yield aldosterone. (B) Aldosterone biosynthesis is shown with the structure of each steroid illustrated.

Figure 4. Angiotensin II (AngII) signaling mediating aldosterone secretion from adrenal glomerulosa cells. AngII binds to the AngII receptor, type 1 (AT1R), which is a G protein‐coupled receptor (GPCR) coupled through the heterotrimeric G protein, G_q/11, to phospholipase C‐β (PLCβ). PLCβ hydrolyzes phosphatidylinositol 4,5‐bisphosphate (PIP₂) to yield inositol 1,4,5‐trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ binds to IP₃ receptors on the endoplasmic reticulum (ER) to release this intracellular store of calcium ions (Ca²⁺). The resulting increase in cytosolic calcium levels activates calcium/calmodulin‐dependent protein kinases (CaMK), which phosphorylates substrates controlling various cellular processes to initiate the steroidogenic response. Diacylglycerol, on the other hand, activates protein kinase C (PKC) isoenzymes, which can activate other effectors such as phospholipase D (PLD), to sustain the aldosterone secretory response. PLD hydrolyzes phosphatidylcholine to generate phosphatidic acid (PA), which can be dephosphorylated by lipid phosphate phosphatases to yield diacylglycerol. The other signal required for sustained aldosterone secretion is an enhanced influx of calcium, which enters the cell through store‐operated calcium influx pathways and voltage‐dependent calcium channels. AngII also depolarizes the glomerulosa cell [i.e., the membrane potential (V _m) is decreased] by inhibiting the activity of potassium channels and activates, through as yet unclear mechanisms, mitogen‐activated protein kinase pathways ending in extracellular signal‐regulated kinase (ERK) and p38, as well as tyrosine kinases, such as Src and Janus kinase‐2 (JAK2). ERK can phosphorylate and activate cholesterol ester hydrolase to cleave cholesteryl esters stored in lipid droplets (LD) and increase free cholesterol available to serve as a precursor for steroidogenesis. On the other hand, Src family kinases, together with PKC, induce the transphosphorylation and activation of protein kinase D (PKD), which phosphorylates and activates activating transcription factor (ATF)/cAMP response element binding (CREB) protein transcription factors to induce the expression of steroidogenic acute regulatory (StAR) protein (see Fig. 6), the early rate‐limiting step in steroidogenesis. CaMK is also able to increase the phosphorylation and activation of ATF/CREB transcription factors. These factors can also stimulate the expression of aldosterone synthase, or CYP11B2, the late rate‐limiting step in aldosterone production. Together, these signal transduction pathways increase the production of aldosterone.

Figure 5. The heterotrimeric G protein cycle. Heterotrimeric G proteins, such as the G_q/11 utilized by the AT1R, are composed of three subunits, α, β, and γ. Activation by G‐protein‐coupled receptors (GPCRs) like the AT1R triggers the α subunit to exchange GDP for GTP, a process that can be accelerated/enhanced by guanine nucleotide exchange factors (GEFs). This then allows the GTP‐bound α subunit to dissociate from the βγ subunits and associate with effector enzymes such as PLCβ. With time, the intrinsic GTPase activity of the α subunit hydrolyzes GTP to GDP, allowing its reassociation with the βγ subunits and terminating G protein‐mediated activation of its effector enzymes. This process can be accelerated by GTPase‐activating proteins (GAPs).

Figure 6. The diacylglycerol‐activated protein kinases, protein kinase C (PKC), and protein kinase D (PKD). Shown is a schematic depicting the PKC isoenzyme and PKD domain structures. The PKC isoenzymes consist of the classical or conventional PKCs, with two cysteine‐rich domains (C1) that bind diacylglycerol (or phorbol esters), a C2 domain that associates with acidic phospholipids like phosphatidylserine upon binding of calcium and the C3 and C4 domains that comprise the catalytic domain and bind substrate (C4) and ATP (C3). Classical PKC isoforms include PKC‐α, ‐βI, ‐βII, and ‐γ and require acidic phospholipids, diacylglycerol, and calcium for full activity. The novel PKC isoenzymes also possess two C1 domains and a C3 and a C4 domain, but instead of a C2 domain, these isoforms possess a C2‐like (C2L) domain that does not require calcium to bind acidic phospholipids. For this reason the novel PKCs, which include PKC‐δ, ‐ϵ, ‐η, and ‐θ, do not require calcium for activity but are activated by acidic phospholipids and diacylglycerol. Atypical PKC isoenzymes have a C3 and C4 domain, no C2 domain and only one C1 domain. These isoforms, which comprise PKC‐ ι/λ and ‐ζ, are unable to bind or become activated by diacylglycerol or phorbol ester and are unresponsive to calcium but require acidic phospholipids. All PKC isoenzymes also possess a pseudosubstrate (PS) domain, not possessed by PKD family members, which include PKD‐1 (also known as PKC‐μ), ‐2 and ‐3 (also known as PKC‐υ). PKD isoforms possess two C1 domains homologous to those in classical and novel PKC isoenzymes (although separated by a longer intervening sequence than in PKC isoenzymes), as well as a catalytic domain composed of a C3 and C4 domain, with the catalytic domain exhibiting more homology to CaMKs than to PKC isoforms. In addition, PKD isoforms have an N‐terminal hydrophobic domain (HD) of unknown function and a pleckstrin homology (PH) domain, which acts as a protein interaction domain.

Figure 7. Protein kinase D (PKD) activation in adrenal glomerulosa cells. PKD is activated by Src family kinase‐induced and PKC‐mediated transphosphorylation in glomerulosa cells. In H295R cells the PKC isoform catalyzing this transphosphorylation is PKC‐ϵ. Also upstream of PKD is the enzyme phospholipase D (PLD), which produces the lipid second messengers phosphatidic acid and diacylglycerol, the latter in conjunction with the activity of lipid phosphate phosphatases. In primary bovine glomerulosa cells, PKD then (whether directly or indirectly) phosphorylates and activates members of the activating transcription factor (ATF)/cAMP‐dependent response element binding protein (CREB) family of transcription factors to increase the transcription of steroidogenic acute regulatory protein (StAR). Increased transcription of StAR, as the rate‐limiting step in acute aldosterone production, allows enhanced aldosterone biosynthesis and secretion. In the H295R cells PKD also stimulates CYP11B2 expression, which as the late rate‐limiting enzyme in long‐term aldosterone production, also increases the synthesis of this steroid.

Figure 8. Sustained AngII‐induced aldosterone secretion requires continued calcium influx. Shown is a schematic illustrating the requirement for continued calcium influx for sustained aldosterone secretion in response to AngII. Whereas under normal physiological conditions AngII elicits a monotonic and sustained increase in aldosterone secretion, in the presence of a calcium channel antagonist such as nitrendipine (NTR) or upon removal of extracellular calcium, AngII induces only a transient increase in aldosterone secretion that returns to basal values over time.

Figure 9. Under physiological conditions, low‐voltage‐activated transient (T)‐type voltage‐dependent calcium channels allow entry of calcium ions via a window current. T‐type calcium channels activate at negative membrane potentials but also inactivate at similarly negative potentials (and deactivate very slowly). However, there is a small range of physiologically relevant membrane potentials at which there is a proportion of T‐type channels that are activated but not yet deactivated or inactivated. In this range calcium current flows generating a “window current.” As shown, AngII shifts the voltage dependence of activation of T‐type channels to more negative values to increase this window current, thus enhancing calcium entry and thereby stimulating aldosterone secretion.

Figure 10. Lipid second messengers involved in AngII‐induced aldosterone production. (A) A schematic illustrating lipid second messengers generated as a result of AngII binding to the AT1R is shown. These include phosphatidic acid (PA), which is generated directly by the action of PLD on phosphatidylcholine or indirectly by phosphorylation of diacylglycerol (DAG) by diacylglycerol kinase, and DAG, generated by PLCβ‐mediated hydrolysis of PIP₂. PA can also be metabolized to DAG by lipid phosphate phosphatases or can be deacylated by phospholipase A to generate lysophosphatidic acid (LPA) and a free fatty acid such as arachidonic acid (AA). In addition, glomerulosa cells express a DAG lipase that preferentially releases AA from DAG; AA can also be released directly from phospholipids by the activity of phospholipase A₂. In turn, AA is metabolized by 12‐lipoxygenase to 12‐hydroxyeicosatetraenoic acid (12‐HETE). (B) The structure of the different lipid molecules and second messengers is illustrated. The phosphatidylcholine shown contains stearic and arachidonic acids (and so generates phosphatidic acid and diacylglycerol containing these fatty acids). Note that different fatty acids in these two positions distinguish different species of each lipid.

Figure 11. Lipid second messengers involved in AngII‐induced aldosterone production. (A) A schematic illustrating lipid second messengers generated as a result of AngII binding to the AT1R is shown. These include phosphatidic acid (PA), which is generated directly by the action of PLD on phosphatidylcholine or indirectly by phosphorylation of diacylglycerol (DAG) by diacylglycerol kinase, and DAG, generated by PLCβ‐mediated hydrolysis of PIP₂. PA can also be metabolized to DAG by lipid phosphate phosphatases or can be deacylated by phospholipase A to generate lysophosphatidic acid (LPA) and a free fatty acid such as arachidonic acid (AA). In addition, glomerulosa cells express a DAG lipase that preferentially releases AA from DAG; AA can also be released directly from phospholipids by the activity of phospholipase A₂. In turn, AA is metabolized by 12‐lipoxygenase to 12‐hydroxyeicosatetraenoic acid (12‐HETE). (B) The structure of the different lipid molecules and second messengers is illustrated. The phosphatidylcholine shown contains stearic and arachidonic acids (and so generates phosphatidic acid and diacylglycerol containing these fatty acids). Note that different fatty acids in these two positions distinguish different species of each lipid.

Figure 12. Potential effectors of the lipid second messengers, phosphatidic acid (PA), and diacylglycerol (DAG). PA can interact with and modulate the activity of a variety of effector enzymes as shown. These include the PIP₂‐synthesizing enzyme phosphatidylinositol 4‐phosphate 5‐kinase (PI4P5K), c‐Raf (a mitogen‐activated kinase kinase kinase upstream of extracellular signal‐regulated kinase), the cAMP‐hydrolyzing enzyme cAMP phosphodiesterase type D (PDE4D3), mammalian target of rapamycin (mTOR) protein kinase, the small GTPase Rac, protein serine/threonine phosphatase 1 (PP1), the protein tyrosine phosphatase SHP‐1, the protein kinase kinase suppressor of ras (KSR), the orphan nuclear receptor steroidogenic factor‐1 (SF1), PKCα, a Ras guanine nucleotide exchange factor (GEF) son of sevenless (SOS), the heterotrimeric G protein subunit G_oα, the potassium channel KcsA, and the protein kinase ribosomal p70 S6 kinase (S6 kinase). Myosin phosphatase and brefeldin A ADP‐ribosylated substrate (not shown) are also reported PA effectors. DAG effectors include the classical and novel PKC isoenzymes, PKD, the Ras GEF Ras guanine nucleotide release protein (RasGRP), the Rac GTPase‐activating proteins chimaerins and the UNC13 proteins, which are involved in vesicle maturation and exocytosis.

Figure 13. Transcription factors regulating CYP11B2 expression in glomerulosa cells. The CYP11B2 promoter possesses several response elements that are bound by transcription factors that regulate the expression of this gene. Transcription factors responsible for controlling CYP11B2 expression include, as illustrated, members of the ATF/CREB family of transcription factors and members of the nerve growth factor‐induced clone B (NGFIB) transcription factor family, such as NGFIB (also known as NR4A1 or Nur77) and nuclear receptor related‐1 protein (Nurr1, also known as NR4A2). Steroidogenic factor‐1 (SF1) appears to exert a biphasic effect on CYP11B2 expression, with transcription decreased both at low and high levels of SF1.

Figure 14. AngII‐induced aldosterone secretion can be mimicked by agents reproducing the calcium and diacylglycerol signals. As schematically illustrated, the importance of both branches of the AngII‐induced signaling pathways resulting from the hydrolysis of PIP₂ is demonstrated by the requirement for two signals to mimic the aldosterone secretory response to this hormone. Thus, an agent that increases cytosolic calcium levels, such as the calcium ionophore A23187, induces only a transient increase in aldosterone secretion. Conversely, an agent that substitutes for DAG in activating its effectors, such as phorbol 12‐myristate 13‐acetate (PMA), elicits a slowly developing rise in aldosterone secretion. On the other hand, the combination of these two agents produces a monotonic increase in aldosterone secretion that resembles the response to AngII. Similarly, increasing calcium influx with the calcium channel agonist BAY K8644 has a minimal effect on aldosterone secretion unless it is added together with synthetic DAGs such as 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG), or DAG‐generating bacterial PLD, to mimic the DAG/PKC signal. In this case the combination induces a monotonic increase in aldosterone production.

Figure 15. AngII can prime adrenal glomerulosa cells to respond to a second AngII exposure to an agent that increases calcium influx with enhanced aldosterone secretion. (A) The ability of AngII to prime zona glomerulosa cells to a second AngII exposure is schematically illustrated. With the priming response, pretreatment of glomerulosa cells with AngII induces an aldosterone secretory response that returns to basal upon removal of AngII (or addition of an AngII receptor antagonist). If the cells are reexposed to AngII within a certain time frame, the second aldosterone secretory response is enhanced relative to the first (or relative to cells that are not pretreated). It should be noted that the window of time differs between freshly isolated cells in a perifusion system (15‐20 min) or cultured cells subjected to frequent medium changes (30‐50 min). (B) AngII pretreatment can also prime glomerulosa cells to respond to agents that increase calcium influx with enhanced aldosterone secretion, relative to cells that have not been pretreated with AngII. Thus, whereas in naïve (unpretreated) cells, the calcium channel agonist BAY K8644 or a slightly elevated extracellular potassium level induces only a minimal aldosterone secretory response, these agents induce substantial aldosterone secretion in AngII‐pretreated cells. Since sustained aldosterone secretion requires two signals, calcium influx and a DAG messenger, the capacity of AngII to induce priming to agents that increase calcium influx suggests that a DAG signal is maintained following AngII removal (or addition of an AngII receptor antagonist) to underlie priming.

Figure 16. Proposed mechanism of AngII‐induced priming. Under control conditions, the AT1R is unoccupied and signal transduction pathways are minimally activated. (A) AngII binding to its AT1R triggers the production of two pools of DAG, a slowly metabolized pool marked by myristate and a rapidly metabolized pool incorporating arachidonate. Because myristate is primarily incorporated into phosphatidylcholine, the substrate of PLD, and AngII activates PLD, the slowly metabolized pool of DAG is likely generated by PLD activity. The combination of the DAG signal and a calcium influx signal stimulates aldosterone secretion. (B) Upon removal of AngII (or antagonism of its receptor), calcium influx rapidly declines as does the arachidonate‐containing DAG, whereas the myristate‐DAG remains elevated, presumably retaining PKC at the plasma membrane. However, in the absence of calcium influx, PKC is inactive or insufficient by itself to induce aldosterone production. (C) Upon re‐exposure to AngII, additional PKC is recruited to the plasma membrane by the small release of ER calcium ions and the extra DAG generated. In addition, calcium influx is reactivated and stimulates PKC activity (or combines with the active PKC) to trigger aldosterone secretion, such that the second AngII exposure induces an enhanced secretory response relative to the first or to cells that are not pretreated with AngII. The PKC maintained at the plasma membrane by the slowly metabolized myristate‐containing DAG following AngII pretreatment also allows the primed cells to respond to agents that activate calcium influx with enhanced aldosterone secretion, relative to those that are naïve (i.e., have had no prior AngII exposure).

Figure 17. Elevated extracellular potassium signaling mediating aldosterone secretion from adrenal glomerulosa cells. Elevated extracellular potassium levels depolarize the glomerulosa cell membrane potential (V _m) by decreasing the electrochemical gradient for potassium efflux through the basal potassium conductance. The small change in membrane polarization activates T‐type calcium channels to increase calcium influx and activate calcium signaling pathways including CaMKs. Elevated potassium levels may also increase cAMP levels by stimulating the activity of a calcium‐sensitive adenylate cyclase (AC), although this effect is somewhat controversial. The increased cAMP levels would then stimulate the activity of cAMP‐dependent protein kinase, or protein kinase A (PKA). Also controversial is whether or not elevated extracellular potassium levels increase phosphoinositide hydrolysis in glomerulosa cells (not shown). An increase in extracellular potassium concentration has also been reported to activate PLD and increase the phosphorylation of myristoylated alanine‐rich C‐kinase substrate (MARCKS), an endogenous PKC substrate, presumably by stimulating PKC activity. The activated CaMK, and possibly PKA, phosphorylates ATF/CREB transcription factors to induce StAR and CYP11B2 expression, the early and late rate‐limiting steps in aldosterone biosynthesis, respectively, thereby stimulating aldosterone secretion.

Figure 18. Adrenocorticotrophic hormone (ACTH) signaling mediating aldosterone secretion from adrenal glomerulosa cells. ACTH binds to the melanocortin receptor (MC2R), a GPCR coupled to adenylate cyclase (AC) through the G_s heterotrimeric G protein. Stimulated adenylate cyclase converts ATP to cAMP, thereby increasing cAMP levels, which in turn stimulate PKA. PKA increases calcium influx to increase intracellular calcium levels and activate CaMK (although one report suggests that the activation of CaMK by ACTH is independent of PKA). PKA and CaMK phosphorylate and activate ATF/CREB transcription factors to induce STAR and CYP11B2 expression, the early and late rate‐limiting steps in aldosterone biosynthesis, respectively; PKA can also phosphorylate and stimulate the activity of StAR. The increase in StAR transcription and phosphorylation, in turn, promotes aldosterone production.