Figure 1. Steroids produced by the adrenal cortex and their respective actions. Free cholesterol is enrolled in three distinct pathways, according to specific enzyme processing: aldosterone in zona glomerulosa; corticosterone (rodents) and cortisol (humans, bovine, ovine, and hamsters) in zonae fasciculata and reticularis; and dehydroepiandrosterone (DHEA) and androstenedione in zona reticularis (mainly in humans, and to a lesser extent in bovine, porcine, and ovine) (153). At the level of the inner mitochondrial membrane, cholesterol is cleaved by the P450 cholesterol side‐chain cleavage enzyme (P450scc) into pregnenolone. Further steps include the enzymes 3β‐hydroxysteroid dehydrogenase type 2 (3β‐HSD2), 17α‐hydroxylase, 17,20‐lyase (P450c17), 21β‐hydrolylase (P450c21), 11β‐hydroxylase (P450c11β), and aldosterone synthase (P450aldo). Steps indicated in blue occur in mitochondria and steps indicated in green in the endoplasmic reticulum. The figure illustrates the physiological role of the three classes of steroids (black boxes) and the pathological consequences associated with an elevated and sustained level of secretion (red arrows and boxes). Adapted, with permission, from Figure 1 (619).

Figure 2. Hematoxylin and eosin (H&E)‐stained sections of adrenal glands from various species of adult mice. (A) C57BL/6J, (B) 129S1/Sv1mJ, (C) CBA/J, (D) BALB/cByJ, (E) A/J, and (F) AKR/J female mice. In all strains, the zona glomerulosa (ZG) is characterized by 3 to 5 layers of small cells, the zona fasciculata (ZF) by cells arranged as centripetal columns separated by sinusoids. On the other hand, the zona reticularis (ZR), localized between the inner zona fasciculata (IZF) and medulla is characterized by smaller cells with irregular dispositions. The X Zone (XZ), consisting of small clumps of loosely distributed cells, is clearly observed in both the 129S1/SvlmJ strain (B) and CBA/J strain (C) and is absent in the A/J (E) and AKR/J strains (F). In female BALB/cByJ (D), A/J (E), and AKR/J (F) mice, the cortex is clearly separated from the medulla (M) by a large lipoid zone (LS). This zone represents up to 50% of the adrenal volume in the A/J strain (E). Scale bar = 100 μm. Adapted, with permission, from Figures 5 and 7 (190).

Figure 3. Morphological impact of gene deletion of Steroidogenic factor 1 (Sf1), Sf1/β‐catenin (A), and Sonic hedgehog (Shh) (B) on adrenal gland organogenesis. (A) Progressive decline of adrenocortical volume in Sf1/Cre^low‐mediated β‐catenin (Sf1/β‐catenin^−/−) mice. Histological analysis of the adrenal glands from wild‐type (WT) and Sf1/β‐catenin^−/− mice at 30 weeks. Adrenal glands were isolated from mice of the indicated genotypes and processed for hematoxylin and eosin (H&E) staining and immunohistochemical detection of Sf1 and β‐catenin. The black bar highlights the adrenal cortex. The adult adrenal gland of SF1‐deficient mice exhibits a reduced size and histological disorganization, with a decreased number of Sf1‐positive cells. Scale bars: 100 μm. C, adrenal cortex; M, adrenal medulla. (B) Abnormal growth and development of the adrenal gland is observed in Shh/SF‐1^−/− mice. Glands from Shh/SF‐1^−/− mice and wild‐type (WT) littermates are compared. 3β‐hydroxysteroid dehydrogenase type 2 (3β‐HSD2) (green) serves as a cortical marker, whereas tyrosine hydroxylase (red) stains the chromaffin cells of the adrenal medulla. Cells are counterstained with DAPI to visualize cell nuclei. At birth (P0), Shh/SF‐1^−/− mice exhibit significant defects in organization of the adrenal cortex and medulla while adrenal mass and cortical thickness are significantly reduced. In adult Shh/SF‐1^−/− mice, the cortex fails to encapsulate the adrenal medulla, resulting in the eccentric position of the medulla. (Scale bars, 200 μm.) Reproduced, with permission, from Figure 7 (378) (A) and Figure 3 (136) (B).

Figure 4. Model of cellular organization and organogenesis of the adrenal cortex. Stem cells or primary progenitor cells putatively arise from the rare undifferentiated adrenocortical cells residing within the capsule (yellow). These cells express shh, but not Sf1 or Dax1, and may be considered as quiescent self‐renewing cells. These primary progenitors subsequently become secondary progenitor cells (green). These cells, under the impulsion of shh, express Wnt and fibroblast growth factor (FGF), which are required for adrenal gland development and maintenance. These subcapsular cells are Sf1 positive and Dax1 positive, transiently amplifying nonsteroidogenic cells (putatively, zona intermedia). Finally, these cells ultimately become mature steroidogenic cells [Sf1 positive, Dax1 negative, as well as Cyp11β2 (P450aldo)‐expressing glomerulosa cells (ZG) (dark blue) or Cyp11β1 (P450c11β)‐expressing fasciculata cells (ZF) (turquoise)]. These cells have a decreased potential for proliferation. Reproduced and adapted, with permission, from Figure 2 (377).

Figure 5. Zonal expression of the steroidogenic enzymes. (A) Pseudocolored images of cytochrome P450 aldosterone synthase (P450aldo, red), cytochrome P450 11β‐hydroxylase (P450c11β, green), and Ki67 (blue) protein, after double immunohistochemical staining in adult rat adrenal glands. Cells stained for P450aldo are localized in the zona glomerulosa (ZG) and separated from P45011β staining in zona fasciculata (ZF) by the nonstained zona intermedia (ZI). Note that not all cells are stained for P450aldo. Scale bar = 100 μm. CP, capsule. (B) Immunohistochemistry for cytochrome b5, DHEA‐sulfotransferase (DHEA‐ST) and 3β‐hydroxysteroid dehydrogenase type 2 (3β‐HSD2) in human adrenal cortex. Both DHEA‐ST and cytochrome b5 immunoreactivities are strongly detected in the cytoplasm of adrenocortical cells in the zona reticularis. Immunoreactivity of DHEA‐ST is weak in the zona fasciculata and undetectable in the zona glomerulosa. Cytochrome b5 is similarly weak in the zonae fasciculata and glomerulosa. On the other hand, reactivity for 3β‐HSD2 is strong in the zonae glomerulosa and fasciculata, but negative in the zona reticularis. Reproduced, with permission, from Figure 2 (216) (A) and from Figure 3 (600).

Figure 6. (A) Impact of the renin‐angiotensin system (A) and of ACTH (B) in adrenal gland proliferation. Depicted are 5‐bromo‐2′‐deoxyuridine (BrdU)‐positive nuclei (red) in the outer cortex (upper panel) and inner cortex and medulla (lower panel) from control rat, Ang II‐infused rat and rat fed a low‐sodium diet (×380). An increase in proliferation can be seen with Ang II and under low‐sodium diet conditions, not only in the zona glomerulosa (ZG), but also in the inner zones of the cortex (inner zona fasciculata, ZF and zona reticularis, ZR), at the junction with the medulla (M). In the presence of a low‐sodium diet, there is no increase in the zona intermedia (ZI). (B) Time‐dependent relationship between steroidogenesis and [³H]‐thymidine incorporation in adrenocortical cells in response to ACTH. Adrenocortical cells from adult rats were plated at a density of 1 × 10⁵ cells per cm². ACTH (1 μg/mL) was added on day 13 of culture, after which steroid production and [³H]‐thymidine incorporation were respectively measured as a function of time. Data points represent the means of analyses of triplicate incubations. An inverse relationship between stimulation of steroidogenesis and inhibition of DNA synthesis is shown. Over a period of 4 days after the addition of ACTH, steroidogenesis is increased while thymidine incorporation is decreased. (C) Dose‐dependent effect of ACTH on steroidogenesis, [³H]‐thymidine incorporation and cAMP concentration in functional adrenal tumor cells. For the steroid assay, cells were grown with the indicated concentrations of ACTH for 24 h after which the amount of corticosteroids released into the medium was assayed. For the determination of [³H]‐thymidine incorporation, cells were incubated with various concentrations of ACTH for 6 hrs. Two hours before the end of incubation, 10 μCi of [³H]‐thymidine was added and the incorporation measured. Each data point of the steroid assay and the determination of [³H]‐thymidine incorporation represents the mean value of five cultures. For the cAMP assay, cells were incubated with ACTH for 30 min and the cAMP content assayed. Each data point represents one determination from five combined cultures. As in panel B, the extent of stimulation of steroid secretion and the amount of inhibition of [³H]‐thymidine incorporation are closely related, in parallel with cAMP content. Reproduced, with permission, from Figures 1 and 2 (484) (A), Figure 2 (605) (B), and Figure 2 (467) (C).

Figure 7. Effect of vasopressin (AVP) on mitotic activity (proliferation) in rat adrenal zona glomerulosa. (A) Long Evans rats were treated with either saline (Control) or AVP (128 μg/day) for 48 h or 6 days. Images are from 5‐μm‐thick paraffin sections stained with hematoxylin and eosin. Magnification ×327. Mitotic activity (metaphasic cells) (arrows) is observed mainly in the most external portion of the zona glomerulosa (ZG), under the capsule but not in the zona intermedia (ZI) or zona fasciculata (ZF). This effect leads to an enlargement of the zona glomerulosa, which is statistically significant after 6 days of treatment. (B) Using hypophysectomized animals, in which the mitotic activity of glomerulosa cells falls drastically, 2‐day treatment with AVP restores this activity by 86%. ACTH treatment is less effective (30%), and when combined with AVP, prevents the effect of AVP. Reproduced, with permission, from Figure 1 (258) and adapted from results of Table 3 (560).

Figure 8. (A) Cellular uptake of cholesterol, intracellular processing and biosynthetic utilization of steroid hormones. Free cholesterol for steroidogenesis can potentially be derived from four different sources. Pathway 1: Cholesterol may be synthesized in the endoplasmic reticulum (ER) from acetyl CoA via the rate limiting enzyme hydroxymethylglutaryl coenzyme A reductase (HMG CoA). The endoplasmic reticulum (ER)‐associated integral membrane protein complex, namely the sterol regulatory element‐binding protein (SREBP) and SREBP cleavage‐activating protein (SCAP), controls the gene expression of a number of enzymes involved in cholesterol biosynthesis including the rate‐limiting enzyme, HMG‐CoA reductase. Pathway 2: Low density lipoprotein (LDL), containing cholesterol, binds to the LDL receptor (LDL‐R), and is trafficked through the endosomal pathway (clathrin‐coated pits, early endosomes, late endosomes, and lysosomes). The transfer of cholesterol to the mitochondria from the late endosomes and lysosomes is facilitated by MLN64, along with the assistance of Nieman‐Pick type C1 (NPC1) and Nieman‐Pick type C2 (NPC2) in transferring cholesterol out of the lysosomes. Pathway 3: Cholesterol may be transferred from high density lipoproteins (HDL) to the plasma membrane or the cytoplasm by the scavenger receptor class B, type I (SR‐BI) receptor, localized in caveolin‐rich domains (called lipid rafts or caveolae). The cholesteryl esterase, called hormone‐sensitive lipase (HSL), converts esterified cholesterol (CE) from the plasma membrane to free cholesterol (FC), which can be used for steroidogenesis. CE can also be stored in lipid droplets (LD). Pathway 4: HSL also interacts with esterified cholesterol present in lipid droplets, generating free cholesterol for use in steroidogenesis. Free (nonesterified) cholesterol (FC) can be esterified for storage in lipid droplets by acyl CoA:cholesterol acyltransferase (ACAT) (step 5) or can be transported into mitochondria for steroidogenesis (step 6). At this step, cholesterol is transferred to the translocator/steroidogenic acute regulatory protein complex (TSPO/StAR complex) for metabolism by the cholesterol side chain cleavage enzyme (P450scc). (B) Schematic representation of the regional composition of the LDL receptor (LDL‐R). The ligand‐binding region consists of seven cystein‐rich repeats (R1‐R7) (green). The second region in the LDL‐R ectodomain consists of three EGF‐like repeats (A, B, and C) comprised of 40 amino acids (illustrated in red and blue), and is involved in preventing the acid‐dependent dissociation of ligand in endosomes. The third region in the LDL‐R ectodomain is enriched in serine and threonine residues that function as acceptor sites for O‐linked sugars (purple). The transmembrane domain consists of a sequence of 24 amino acids which anchors the LDL‐R in the lipid bilayer. Endocytosis and intracellular transport of the LDL‐R are regulated via its cytosolic domain. The NPXY motif adopts a tight hairpin conformation that serves as a binding site for a variety of adaptor proteins and signaling molecules, including clathrin. (C) Schematic representation of the scavenger receptor B type I (SR‐BI): (1) cholesterol flux (orange circles) is facilitated by selective proteins, such as CLAMP (carboxy‐terminal linking and modulating protein); (2) the C‐terminal PDZ‐interacting domain of SR‐BI (including K509), mediates direct interaction with adaptor molecules; and (3) the C‐terminal transmembrane domain (CTTM) of SR‐BI is required for signaling. Cholesterol binds directly to the CTTM. ApoA‐I indicates apoliporotein A‐I; HDL, high‐density lipoprotein. Reproduced, with permission, from Figure 3 (268) (B) and Figure 1 (641).

Figure 9. Steroidogenesis in the adrenal gland. Following the StAR‐mediated uptake of cholesterol into mitochondria of adrenocortical cells, aldosterone, cortisol, and adrenal androgen precursors are synthesized through the coordinated action of a series of steroidogenic enzymes in a zone‐specific manner. The adrenal cortex produces zone‐specific steroids as a result of the differential expression of steroidogenic enzymes. In the initial step of steroidogenesis, steroidogenic acute regulatory (StAR) protein is needed for the rate‐limiting step of movement of cholesterol to the inner mitochondrial membrane, where cholesterol is cleaved by cholesterol side‐chain cleavage (P450scc) to pregnenolone. Further steps of the steroidogenic pathway include the enzymes 3β‐hydroxysteroid dehydrogenase type 2 (3β‐HSD2), 17α‐hydroxylase, 17,20‐lyase (P450c17), 21‐hydrolylase (P450c21), 11β‐hydroxylase (P450c11β), and aldosterone synthase (P450aldo). Adapted, with permission, from Figure 1 (26).

Figure 10. Schematic representation of proopiomelanocortin (POMC) processing and importance of selected ACTH amino acids. (A) Prohormone convertase 1 (PC1 also called PC3) catabolizes the parent POMC peptide into three families of peptides, including the ACTH‐peptide family (green), the β‐lipotropin peptides (pink), and the N‐terminal POMC peptides (blue). The ACTH sequence is further cleaved (by PC2) to generate α‐MSH and the corticotrophin‐like intermediate peptide (CLIP); the C‐terminal β‐LPH is further cleaved to generate γ‐LPH, β‐endorphin and β‐MSH, while the N‐terminal POMC (also named Pro‐γ‐MSH) can be cleaved into N‐POMC (1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52), which in turn can be further cleaved into N‐POMC (1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28) by the adrenal secretary protease (Asp). On the other hand, the second product of Pro‐γ‐MSH cleavage is γ3‐MSH, which may be further cleaved in γ2‐MSH and γ1‐MSH. The final products are generated in a tissue‐specific manner. (B) Functional domains in the ACTH sequence. The amino acid sequence highlighted in red (HFRW) (His⁶‐Phe⁷‐Arg⁸‐Trp⁹) is essential for binding and cAMP production by α‐MSH. A second sequence highlighted in green (KKRR) (Lys¹⁵‐Lys¹⁶‐Arg¹⁷‐Arg¹⁸) is essential for the binding of ACTH to its receptor. The first sequence HFRW is named “message,” while the second sequence KKRR is called “address.” Certain fragments, such as ACTH (11,12,13,14,15,16,17,18,19,20,21,22,23,24) and ACTH (7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39) not only lack activity, but act as competitive antagonists of the effect of full length ACTH. Reproduced, with permission, from Figure 2 (253).

Figure 11. Importance of melanocortin‐2 receptor accessory proteins (MRAPs) in ACTH/MC2R expression and functionality. (A) Stable isogenic 293/FRT cells expressing either MRAPα or MRAPβ were transiently transfected with Myc‐hMC2R or MC2R‐GFP and assessed for cAMP production in the presence of 1 μmol/L IBMX for 15 min. Upper panel: the data are expressed as fold increases over basal cAMP levels to illustrate maximal effects. Lower panel: the data from upper panel were normalized from 0% to 100% to visualize shifts in dose‐response curves. Maximal cAMP stimulation is lower in MRAPα‐expressing cells, although normalization indicates that MRAPα‐expressing cells are more efficient. In MC2R‐GFP transfected cells, responses are lower. When illustrated as normalized data (lower panel), EC₅₀ values for MC2R‐GFP expressed in MRAPα and MRAPβ isogenic cell lines are clearly right shifted compared with untagged‐ and Myc‐MC2R cell lines. Thus, the presence of GFP at the C terminus of MC2R alters the potency and sensitivity of the ACTH response (635). (B) Immunofluorescence labeling of MRAP isoforms and of MC2R expressed in double stable isogenic cell lines. MRAPβ with anti‐Flag antibody (Ba), MC2R was labeled with anti‐c‐Myc antibody (Bb) and subsequently detected using secondary antibodies coupled to Alexa‐Fluor594 for MRAP (red) and to Alexa‐Fluor488 for Myc‐MC2R (green). Green and red signals were acquired at the nuclear plane to visualize both the outside and cytoplasmic border of the cell membranes. Both MRAPβ and hC2R are localized at the cell membrane. However, as shown in the merged image (Bc) and magnifications (Bd), red and green fluorescent labeling are not always superimposed. Magnifications clearly evidence MRAPβ at the cytoplasmic (cyt) face of the plasma membrane, while MC2R is clearly outside‐oriented (out), with both interacting partially. Images are representative illustrations of at least 100 cells from three separate experiments. Scale bar, 10 μm (a‐c) or 2.5 μm for magnifications. Reproduced, with permission, with some modifications from Figures 3 and 7 (636).

Figure 12. Role of intracellular Ser and Thr residues in MC2R cell‐surface expression. (A) Representation of the human MC2R depicting the Ser/Thr amino acid residues which have been mutated in the results presented in panel B. (B) Individual 293/FRT/Myc‐mutant cell lines transiently transfected with MRAPβ were submitted to a time‐course challenge with 100 nmol/L ACTH, then processed for determination of cAMP measurements (a‐c) or cell‐surface Myc‐tagged receptors by ELISA (d). As illustrated, mutations of T131, T143, and T147 into either A or D have major repercussions on cAMP accumulation and MC2R functional expression. Indeed, cell surface expression of these mutants are either drastically impaired (for T131) or abrogated (for T143 and T147) [thus, not illustrated in panel (d)]. These results point mostly to the second intracellular loop as being crucial for MC2R expression and functional regulation. Reproduced, with permission, from Figure 5 (253).

Figure 13. Structure‐activity relationships. Steroidogenesis and accumulation of cyclic AMP in response to various lengths of ACTH fragments. (A) Binding affinity and (B) potency of truncated ACTH peptides in OS3 cells stably transfected with wild‐type hMC2R. (A) OS3 cells transfected with hMC2R were incubated with ¹²⁵I‐ACTH in the presence of the indicated amounts of unlabeled ligands after which total ¹²⁵I‐ACTH binding was determined. (B) Cells were incubated with the indicated amounts of peptides and total cAMP accumulation was determined using a competitive binding assay, as described in (126). The EC₅₀ values were respectively 8.59 ± 0.65 nmol/L for ACTH (1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39), 2.8 ± 0.5 nmol/L for ACTH (1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24), 46.7 ± 8.5 nmol/L for ACTH (1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17) and 567 ± 34 nmol/L for ACTH (1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16). (C) Corticosterone production by aliquots of a suspension of rat isolated adrenal cells in response to ACTH (1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39) alone and in combination with 100 μg ACTH (11,12,13,14,15,16,17,18,19,20,21,22,23,24). The values represent quantities in aliquots to which ACTH was added minus quantities in aliquots to which vehicle only was added. Aliquots were incubated for 60 min at 37°C. (D) Effects of various ACTH fragments on cyclic AMP (cAMP) production in cultured Y1 mouse adrenocortical cells. Cells were cultured in a medium containing 10% fetal calf serum, and were stimulated for 1 h with the indicated ACTH fragments. Cyclic AMP was measured by radioimmunoassay. A significant increase in cAMP can be observed with ACTH (l‐39) and ACTH (l‐24), both used at the concentration of 100 nmol/L. The concentration of ACTH (11,12,13,14,15,16,17,18,19,20,21,22,23,24) was 10 μmol/L; that of ACTH (1,2,3,4,5,6,7,8,9,10) and ACTH (11,12,13,14,15,16,17,18,19,20,21,22,23,24) was 100 μmol/L. As can be seen, the combination of ACTH (l‐39) and ACTH (l‐24) with ACTH (11,12,13,14,15,16,17,18,19,20,21,22,23,24) significantly decreases the responses of ACTH (l‐39) and ACTH (l‐24) alone. ^***, P < 0.001. Ctl, control. (E) ACTH (7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38) is devoid of corticosteroidogenic activity but inhibits corticosterone production stimulated by ACTH (1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39) in isolated rat adrenal cells (by 95% when used at 10‐fold molar concentration). This is the only known analogue from a natural source capable of antagonizing the adrenal‐stimulating activity of ACTH. Thus, ACTH (7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38) may be designated corticotropin‐inhibiting peptide (CIP). Reproduced, with permission, from Figure 2 (126), Figure 2 (665) and adapted from Table 3 (427) and adapted from Table 1 (503) (C).

Figure 14. Role of adenylyl cyclases (ACs), phosphodiesterases (PDEs), and phosphatases (PTPs) in ACTH signaling. According to the data published to date, ACTH putatively stimulates AC5/AC6 isoforms which, through cAMP and PKA, activate a slow but sustained Ca²⁺ influx through L‐type channels. Calcium and cAMP interact closely through positive feedback loops to enhance steroid secretion. Thereafter, Ca²⁺ activates the AC3 isoform. Studies have also shown that cAMP can bind cAMP‐GEFs which activate the exchange factors, thereby allowing Epac1 and Epac2 to catalyze the exchange of GTP for GDP on Rap GTPases. The GTP‐bound forms of Rap activate multiple cellular functions, including ERK1/2 and ionic channels (green pathway). Moreover, pertussis toxin (PTX) treatment indicates that ACTH is not only linked to G_s, but also to G_i protein. Activation of αi contributes to the inactivation of the AC3 isoform, although the release of βγ can also activate the AC2 isoform. Meanwhile, βγ‐subunits may stimulate other effectors such as phospholipase Cβ3 isoform, MAPK cascade or cationic Cl⁻ channels. Phosphodiesterases (PDEs) degrade cAMP in such a way that they can precisely control the level of intracellular cAMP. In adrenocortical cells, several isoforms of PDE have been described. In particular, ACTH induces a rapid, but transient inhibition of PDE2, thus maintaining elevated cAMP levels. In fasciculata cells, the PDE8 family is also shown to modulate the cAMP pool under basal to minimally stimulated conditions. When cAMP is more highly stimulated, other IBMX‐sensitive PDEs are activated to restore the basal level of cAMP (purple pathway). On the other hand, PKA leads to activation of phosphotyrosine phosphatases (PTPs) with the subsequent dephosphorylation of specific substrate(s) (still unknown). These proteins potentially directly or indirectly control the induction of an acyl‐CoA synthetase (ACS4), a key enzyme in the stimulation of steroidogenesis. This ACS4 sequesters arachidonic acid (AA) as arachidonyl‐CoA (AA‐CoA). The action of a specific thioesterase, Acot2, allows the release of AA in a specific compartment of the cells. Free AA acts as an inductor of StAR protein and steroidogenesis (pink pathway). AA can also be released from a direct effect of ACTH on PLA₂ (blue pathway). Finally, studies have shown that ACTH is able to stimulate phosphoinositide breakdown; however, the production of inositol trisphosphate induced by ACTH is not sufficient to release calcium from intracellular stores, rather suggesting a role for diacylglycerol (DAG) and protein kinase C (PKC) (gray pathway).

Figure 15. G protein‐dependent (A) and G protein‐independent (B) signaling pathways induced by Ang II binding to AT₁R in adrenal zona glomerulosa. (A) The G protein‐dependent “first wave of signaling,” which includes immediate and secondary responses. Ang II binding to the AT₁R in the adrenal zona glomerulosa induces an immediate coupling to G_αq, resulting in activation of phospholipase C‐β1 (PLC). The latter catalyzes the breakdown of the membrane lipid phosphatidylinositol‐4,5‐bisphosphate (PIP₂) into inositol triphosphate (InsP₃) and diacylglycerol (DAG), which, respectively, act to increase cytosolic Ca²⁺ concentrations and activate PKC (pink pathway). In a more or less simultaneous manner, Ang II induces cell depolarization, inhibiting voltage‐dependent potassium (K⁺) channels, which triggers activation of T‐ and L‐voltage‐dependent calcium (Ca²⁺) channels. In the cytoplasm, Ca²⁺ activates calcium/calmodulin‐dependent protein kinase I/II (Ca²⁺‐CaM kinase) (orange pathway). As secondary G‐dependent pathways, Ang II also activates phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA), which can be metabolized to DAG by lipid phosphate phosphatases. Finally, as in the case of ACTH, Ang II can activate a PLA₂ which generates the release of arachidonic acid (AA) (blue pathways). DAG activates various PKC isoforms as well as protein kinase D (PKD). These mediators, in particular PKC, can then activate the extracellular signal‐regulated kinase (ERK1/2). DAG can be hydrolyzed by DAG lipase to release arachidonic acid (AA), which can be further metabolized by 12‐lipoxygenase to 12‐hydroxyeicosatetraenoic acid (12‐HETE) (blue pathway). Likewise to ACTH, various protein tyrosine phosphatases are also involved in Ang II action. These proteins can directly or indirectly control the induction of an acyl‐CoA synthetase (ACS4), a key enzyme in the stimulation of steroidogenesis. This ACS4 sequesters arachidonic acid (AA) as arachidonyl‐CoA (AA‐CoA). The action of a specific thioesterase, Acot2, allows the release of AA in a specific compartment of the cells. Free AA acts as an inductor of StAR protein and steroidogenesis. (B) G protein‐independent “second wave of signaling,” which includes secondary as well as chronic responses to Ang II. Firstly, as illustrated in the left portion of the figure (illustrated in purple), AT₁R induces activation of the JAK/STAT pathway. Secondly, Ang II can induce signaling pathways leading to ERK1/2, either through various PKC isoforms, generated from PLD activation (illustrated in blue), Src, extracellular matrix/cytoskeleton [in particular the focal adhesion kinase (FAK) and paxillin (PAX)] or through AT₁R‐mediated transactivation of receptor tyrosine kinases (RTKs) such as EGFR. In addition, AT₁R desensitization (induced by cytoplasmic serine/threonine phosphorylation of AT₁R) enhances binding of β‐arrestin to the cytoplasmic tail of AT₁R to inhibit G_αq coupling to AT₁R. β‐arrestin serves as a scaffold for signaling effectors such as Src, resulting in downstream activation of cytoplasmic ERK. All these signaling cascades lead to activation of the mitogen‐activated protein kinase (MAPK) pathway, which includes extracellular‐signal‐regulated kinase (ERK), Jun N‐terminal kinase (JNK) and p38 isoforms (right portion of the figure, shown in green).

Figure 16. Molecular determinants of the functional selectivity and pharmacological applications of the angiotensin II type 1 receptor (AT₁R). (A) Representation of the rat AT_1A receptor, showing selected residues known to be important for signaling. The following color coding is depicted: Blue: selected residues involved in Ang II binding. Green: residues of specific importance to G protein coupling. Light green: residues implicated in G protein coupling with no other outcomes tested. Orange: sites established to be phosphorylated and favor β‐arrestin recruitment. Yellow: residues important for JAK2 stimulation. Magenta: truncation leads to enhanced G protein coupling. Light orange: truncation abrogates Src activation. In addition, the C‐terminus of AT₁R directly and strongly binds to tubulin. Mutation that disrupts tubulin binding dramatically inhibits the cell surface expression of AT₁R, which remains localized in the endoplasmic reticulum (763). (B) Pharmacological potential of biased agonism at the AT₁ receptor. Left panel, Ang II binds the AT₁ receptor to activate both G protein‐dependent and independent signaling. These mechanisms increase acute and secondary steroidogenesis and steroid secretion, as well as chronic effects on growth and transcription of steroidogenic enzymes. Binding of a conventional AT₁ receptor antagonist blocks all effects of AT₁ receptor signaling. Right panel, Application of a “biased ligand” (which has the ability to selectively stimulate a subset of a receptor's activities), may enable selective blockade of detrimental G protein‐dependent responses while activating G protein‐independent signaling. The relative impact of G protein‐dependent and independent mechanisms on metabolic regulation remains to be established. Current therapeutic modalities block both beneficial and detrimental effects. Biased ligands such as SII‐Ang II (336) may allow pathway‐specific targeting of receptor actions and hold promise for future therapeutic options. Reproduced, with permission, from Figure 2 (22) (A), Figure 1 (728) (B).

Figure 17. Expression of angiotensin II receptors in the adrenal gland. (A) Autoradiography of total Ang II receptors (radioactive ligand alone), AT₁ receptors (after blockade of AT₂ receptors with 10 μmol/L PD123177, an AT₂ receptor antagonist), and AT₂ receptors (after blockade of AT₁ receptors with 10 μmol/L Losartan, an AT₁ receptor antagonist) in rat kidney and adrenal glands at postnatal day 2. (B) Ang II AT_1A, AT_1B, and AT₂ receptor mRNA in the adrenal zona glomerulosa and adrenal medulla. Scale bar = 1 mm. In adrenal glands from fetuses and newborns, the AT₂ receptor is more abundant than the AT₁ receptor. Thereafter, its expression decreases, but remains abundant. In the adult adrenal gland, both AT₁ and AT₂ receptors are present in adrenal zona glomerulosa while only the AT₂ receptor is expressed in the medulla. (C) Effect of bilateral nephrectomy in rats on the expression of the AT₂ receptor and CYP11B2 mRNA in the adrenal cortex revealed by nonradioactive in situ hybridization. Representative sections of adrenal glands of a sham operated group (upper panel) and nephrectomy group (lower panel). Compared to sham‐operated rats (a), bilateral nephrectomy leads to a marked increase in the expression of AT₂ receptor mRNA in the zona glomerulosa (ZG) (c). Concomitantly with the enlargement of the steroidogenic active zona glomerulosa (ZG) (from 2 or 3 cell layers to 5 or 6 cell layers), as defined by the expression of CYP11B2 mRNA (b and d), the area of cells expressing AT₂ receptor mRNA increases similarly from 2 or 3 layers to 5 to 7 layers of cells. However, in contrast to the homogeneous distribution of CYP11B2 in bilateral nephrectomy animals, distribution of AT₂ receptor mRNA is not homogeneous. Only a few clusters of cells are found to express the AT₂ receptor gene. Reproduced, with permission, from Figure 2 (9) (A), Figure 8 (425) (B) and Figure 2 (576) (C).

Figure 18. Effects of ACTH (A) and angiotensin II (Ang II) (B) on intracellular calcium concentration (Cai) in adrenal glomerulosa cells. Microfluorometric measurements of Cai were recorded in individual cells loaded with the fluorescent intracellular indicator Fura2/AM, as described in (711). Briefly, cells were cultured on coverslips, loaded with Fura 2/AM, placed in a perfusion chamber, and mounted on an inverted Nikon microscope. The test solutions were applied near the tested cell using a micropipette positioned by a micromanipulator (with a superfusion rate of 1 mL/min). Ca²⁺‐free solutions were obtained by adding 0.5 mmol/L EGTA in a HBS buffer solution. Cai was recorded with a Fluoroplex II system and is illustrated as the ratio of fluorescence at two excitation wavelengths (340 and 380 nm) with the resulting emission collected at 510 nm. (A) Effect of ACTH on Cai response in bovine glomerulosa cells: (a) The Cai response induced by 1 pmol/L ACTH appears very slowly. A progressive increase in Cai is seen, followed by a plateau which falls abruptly after washing with ACTH‐free control medium (a) or by adding EGTA (b). Addition of 50 μmol/L Bay K 8644 (a L‐type Ca²⁺ channels activator) leads to an immediate and further increase in Cai while the subsequent removal of extracellular calcium (c) or addition of the calcium channel blocker nifedipine (10 μmol/L) (d) abolishes the Cai increase induced by ACTH. Finally, likewise to ACTH, 8‐bromo cAMP (8‐Br‐cAMP) enhances Cai (e), while addition of the PKA inhibitor HA1004 abrogates the ACTH response (f). (B) Illustration of Cai recordings in single bovine adrenal glomerulosa cells: (a, b) In these cells superfused with medium containing 1.1 mmol/L Ca²⁺, addition of Ang II (100 nmol/L) induces a rapid increase in Cai followed by a sustained plateau. Removal of Ca²⁺ from the superfusion medium (a) or addition of 10 μmol/L nifedipine (b) induces a rapid decline in the plateau and a return to baseline values. (c) In this cell superfused with Ca²⁺‐containing medium, Ang II (100 nmol/L) induces rapid oscillations which quickly cease when the cell is superfused with a Ca²⁺‐free medium. (d) In this example, the cell is superfused with a Ca²⁺‐free medium during 10 min prior to Ang II application (100 nmol/L). A single peak of Cai rapidly returns to baseline values. Reproduced, with permission, from Figures 3, 6 and 7 (711).

Figure 19. Electrophysiological properties of zona glomerulosa cells. The resting membrane potential is maintained as a result of background K⁺ channels of the KCNK gene family (the TWIK‐related acid‐sensitive K⁺ (TASK) channel TASK‐1, TASK‐3, and bTREK‐1). Glomerulosa cells also express various types of voltage‐dependent K⁺ channels and two types of voltage‐dependent calcium channels (T‐type and L‐type Ca²⁺ channels) which promote Ca²⁺ influx when open. Under resting conditions, the intracellular calcium concentration (Cai) ranges from 50 to 150 nmol/L, according to cell types. Exchangers and Ca²⁺‐permeant nonselective channels, such as chloride channels, also participate in the maintenance of basal Cai. Binding of ACTH stimulates adenylyl cyclase‐induced cAMP and PKA activation. Thereafter, PKA phosphorylates and activates a slow but sustained Ca²⁺ influx through L‐type channels. On the other hand, Ang II, through Gq stimulation of phospholipase Cβ and phosphoinositide breakdown, generates diacylglycerol, an activator of protein kinase C (PKC), and inositol 1,4,5‐trisphosphate (InsP3) which releases Ca²⁺ from intracellular stores near the mitochondria. Under these stimulated conditions, Cai can rise to 200 to 600 nmol/L. In the cytoplasm, Ca²⁺ activates calcium/calmodulin‐dependent protein kinase (Ca²⁺‐CaMK) as well as certain protein kinase C isoforms (PKCs). The subsequent depletion of Ca²⁺ stores activates CRAC channels responsible for the capacitative Ca²⁺ influx. This additional influx allows the refilling of intracellular stores. Finally, exchangers (Na⁺‐Ca²⁺) and pumps (Na⁺/K⁺ and Na⁺‐K⁺‐ATPase) participate in the trafficking of Ca²⁺ across the plasma membrane.

Figure 20. Mislocalization of aldosterone synthase in adrenal glands of Task1^−/− mice is sex‐dependent. (A) In adult Task1^+/+ mice of either sex and in male Task1^−/− mice, aldosterone synthase staining is restricted to zona glomerulosa cells. In female Task1^−/− mice (upper right panel), regular aldosterone synthase staining is disrupted and broadened to the inner areas of the adrenal cortex. The right portion of each panel shows higher magnification images of the adrenal cortex (aldosterone synthase in green, differential interference contrast in gray scale, nuclear staining with HOE33342 in blue). (B) Effects of castration, estradiol (estr.), and testosterone (test.) treatment on aldosterone synthase localization. Male mice were castrated at the age of 5 weeks, followed by treatment with or without estradiol benzoate (4 mg/g/day, s.c., for 5 weeks). In male Task1^−/−, castration (with or without estradiol treatment) does not affect adrenocortical zonation. Sham‐operated male Task1^−/− also show normal zonation (upper panel). In male Task1^−/−, castration prevents normal zonation. Estradiol appears to reduce aldosterone synthase expression without having clear effects on zonation patterns. In female Task1^−/− mice, treatment with testosterone for 3 weeks induces normal zonation, highlighting the importance of androgens for adrenocortical rezonation in Task1^−/− mice (lower panel). Reproduced, with permission, from Figure 4 and 7 from (317).

Figure 21. Effect of ACTH (A) and Ang II (B) on K' currents. (A) Effects of ACTH on outward currents. (a) ACTH was applied at three different concentrations. In each case are illustrated control (trace 1), effect of ACTH (trace 2) and recovery after washout (trace 3). Holding potential, −65 mV, test potential, 20 mV. Calibrations: horizontal, 50 ms (ACTH, 0.1 and 1 nmol/L) and 40 ms (ACTH, 10 nmol/L); vertical, 200 pA (ACTH, 0.1 and 1 nmol/L) and 250 pA (ACTH, 10 nmol/L). (b) The slow outward current is not significantly blocked; the higher trace represents the control, the middle and lower traces are ACTH 10 nmol/L, applied for 2 and 5 ms, respectively; Holding potential, −65 mV; test potential, 70 mV. (c) Differential effect of ACTH on transient and slow outward currents in the same cell. Four traces recorded at voltages indicated at the left of each recording before (circle) and after (squares) application of ACTH (10 nmol/L; 5 min). By blocking the transient outward current, ACTH reveals the slow outward current. (d) The difference between the two traces (before ACTH minus after ACTH) is the current blocked by ACTH. This current is very similar to the transient outward current. Calibrations: horizontal, 250 ms; vertical, 100 pA. (B) Effects of Ang II on outward currents from human zona glomerulosa cells. (a) Upper panels: whole cell current from 2 representative cells recorded from a holding potential of −90 mV; pulses from −110 to 90 mV (cell 1) and 60 mV (cell 2) are provided. Traces represent current recorded during a 90 mV step before, after 5 min of Ang II application, and 5 to 15 min after removal of Ang II. As can be seen, Ang II decreases the outward current. Reversal could be demonstrated for cell 1. Voltage‐clamp steps were applied in 10 mV increments. Standard bath and pipette solutions were used, and Ang II (10 nmol/L) was applied by puffer superfusion for 10 min; (b) lower panels: full current/voltage relation before, during and after Ang II application. Reproduced, with permission, from Figure 5 (556) (A) and Figure 4 (89).

Figure 22. Effect of ACTH and Ang II on Ca²⁺ currents. (A) T‐component voltage characteristics, illustrated for a rat glomerulosa cell. The cell was bathed in a 20 mmol/L Ca²⁺ medium. (a) Currents obtained at various voltages from a holding potential of −80 mV. The current activates and inactivates rapidly. (b) Current‐voltage relationships measured at the peak. The threshold voltage is −60 mV, the maximum current is obtained at −30 mV and the zero‐current voltage is +35 mV. (B) L‐component voltage characteristics. The cell was superfused with a 20 mmol/L Ca²⁺ solution. (a) The current begins to be activated at voltages around −20 mV. In this particular cell, after an activation phase, the current is sustained. (b) Current‐voltage relationships for the L‐component. In a Ca²⁺ medium, the voltage of the maximum current is found at +60 mV and the zero‐current voltage can be estimated to be more positive than + 100 mV. (C) Effects of ACTH on the T‐ and L‐currents. The holding potential was −80 mV, and voltage step depolarizations up to −10 mV were applied. (a) Trace 1 corresponds to the control current. Traces 2 and 3 are current traces obtained after superfusion with 10 nmol/L ACTH‐containing medium. Trace 4 corresponds to the current recorded 4 min after the control medium was resuperfused. (b) Time course of the increase in L‐current amplitude induced by ACTH. The current amplitude was measured at the end of the 800‐ms voltage step depolarizations before, during and after superfusion of the medium with 10 nmol/L ACTH. The time and the duration of the superfusion are indicated by the horizontal bar. Current amplitudes were normalized to the control current amplitude. Numbers (1,2,3,4) refer to the traces in A; the ratios I/I_Control are 2.09, 4.26, 11.23, and 13.32, respectively. (D) Ang II modulation of voltage‐gated Ca²⁺currents measured using perforated‐patch clamp. Voltage‐gated Ca²⁺ current evoked by ramp voltage commands from −128 to 52 mV at a rate of 0.6 mV/ms. Ang II (10 nmol/L) inhibits the second peak of inward current measured at approximately 0 mV within 3 min after the onset of stimulation. L‐type Ca2+ current measured as the current at the end of a 100 mS square wave voltage command to +7 mV following a 4 s prepulse to −108 mV. In this example, stimulation with 10 nmol/L Ang II for 3 min reduces the L current by 50% without affecting the amplitude or inactivation kinetics of the T current component. The holding potential was −88 mV. Reproduced, with permission, from Figure 2 and 3 (210) (A and B) and Figure 8 (441) (C).

Figure 23. Proposed model of interactions between the Ang II AT₁ receptor (AT1‐R), the extracellular matrix (ECM), integrins, and the cytoskeleton in adult rat glomerulosa cells. Implication in proliferation and protein synthesis. (A) In control conditions, binding of fibronectin or collagen to integrins promotes solid adhesion of the cells. In these conditions, cells have a flattened polygonal morphology, characterized by a discrete network of thin stress fibers crossing the entire cell and the presence of focal adhesion points evidenced by paxillin labeling, as illustrated by the green fluorescent dots at the membrane level. On the other hand, paxillin and focal adhesion kinase (FAK), induce specific activation of actin‐associated kinase, RhoA/ROCK. The latter dictates actin cytoskeleton (stress fiber formation) and signaling pathways (such as p42/p44^mapk), leading to basal cell proliferation and steroid secretion (pathway in green, right portion of A). Ang II induces a rapid but transient formation of an intense F‐actin ring at the cell membrane, a disruption of the stress fiber network, and the formation of several thin filopodia in lieu of focal adhesions (cell illustrated in the left portion of panel (A). These changes are accompanied by a disappearance of paxillin labeling at the membrane level and activation of Rac and p38 MAPK. During Ang II stimulation, p42/p44 ^mapk is activated but also requires interaction with p38 MAPK to fully increase cell protein content (pathways in pink). (B) Effect of MAPK inhibitors on angiotensin II‐induced expression of StAR and 3β‐HSD. Glomerulosa cells were cultured for 3 days without or with PD98059 (10 μmol/L) (an inhibitor of MEK) or with SB203580 (10 μmol/L) (an inhibitor of p38 MAPK) introduced 30 min prior to Ang II (5 nmol/L) stimulation. Following hormonal stimulation in the culture medium, cells were processed for Western blot analyses. As can be seen, Ang II increases the expression levels of StAR and of 3β‐HSD which are suppressed in cells preincubated with PD98059 (10 μmol/L) or SB203580 (10 μmol/L). These results indicate that p42/p44^mapk and p38 MAPK play a key role in Ang II‐stimulated aldosterone production by enhancing expression of StAR and 3β‐HSD proteins. Adapted, with permission, from Figure 2 (539) (A) and Figure 3 (545) (B).

Figure 24. Cell to cell communications through gap junctions. (A) In this example, ACTH, through cAMP, activates cAMP‐dependent protein kinase (PKA) in coupled cells via gap junctions (GJ). In this way, stimulation in one cell enables the propagation of stimulation to neighboring cells, thus increasing the cell's response (for ACTH, an increase in steroid production with a concomitant decrease in cell proliferation). (B) In addition, ACTH acting via cAMP is thought to increase the number of available open gap junction channels in a gap junction plaque. It should be noted that in this illustration, one of the cells lacks ACTH receptors, but is able to respond as observed for the MC2R‐expressing cell. Reproduced, with permission, from Figure 6 (512).

Figure 25. Schematic representation of the proposed mechanism of action of Seladin‐1 in adrenocortical cells. (A) Seladin‐1, also named 24‐dehydrocholesterol reductase (DHCR24), is involved in the late steps of cholesterogenesis (8), since it is known to catalyze the conversion of desmosterol to cholesterol (737). In humans, mutations of the DHCR24 gene result in a rare and severe recessive autosomic disorder called desmosterolosis. This pathology is characterized by desmosterol accumulation in plasma and tissues, by multiple congenital anomalies, and by severe mental retardation. (B) Seladin‐1 may play a dual role in the regulation of steroidogenesis and in the protection of adrenocortical cells against negative side‐effects resulting from intense steroidogenesis. Indeed, under control condition, basal steroid production is not significantly affected by Seladin‐1 localized in the cytoplasm. However, under ACTH stimulation, there is activation of the cholesterol biosynthetic pathway, with possible accumulation of reactive oxygen species, (ROS), lipid peroxidation and reactive aldehyde metabolites, all of which generate important oxidative stress. In this instance, nuclear Seladin‐1 may be involved in the protection of adrenocortical cells, due to its ability to bind both the tumor suppressor p53 and the E3‐ubiquitin‐ligase Mdm2, and its ability to displace Mdm2 from p53. Seladin‐1 protects p53 from Mdm2‐induced degradation thus enabling its accumulation. By modulating p53‐Mdm2 interplay, nuclear Seladin‐1 may thus adapt the cell's responses to various stressors, including metabolic stress, these cells having to constantly choose between adaptation to stress or apoptosis. Reproduced and adapted, with permission, from Figure 4 (539).

Figure 26. Schematic illustrations of putative selective signaling platforms which may implicate protein kinase C (PKC), A kinase‐anchoring proteins (AKAPs), various isoforms of adenylyl cyclases (ACs), phosphodiesterases (PDEs), and other scaffolds. AKAPs can localize many signaling proteins in specific locations within the cell, creating preferential interactions on the scaffold (AKAPs can increase the rate at which signal transduction occurs or increase the magnitude of the signal response). Similar platforms and interaction in the adrenal gland could be anticipated from the information provided in many studies, For example, it is known that AKAP79/150 can associate with K⁺ voltage‐dependent channels (such as KCNQ2), together with protein phosphatase‐1 (PP1) or PP2A (a) and PKC; AKAP79/150 can link adenylyl cyclases with protein kinase A and L‐type Ca²⁺ channels, creating a particular platform of signaling (b); AKAP350 has been shown to associate with ACs, and a cAMP‐specific phosphodiesterase, PDE4D3 (c). AKAP79 or other specific AKAPs can interact with PKC (d), Rho, Rac and cytoskeleton (e). Studies have shown that the link between hormone‐induced minimal cAMP levels and activation of cholesterol transport necessary for steroid synthesis, at the level of mitochondria, involves a protein called peripheral‐type benzodiazepine receptor (PBR)‐associated protein (PAP7) (f). According to the authors, this protein functions as an AKAP, critical in cAMP‐dependent steroid formation (434).

Figure 27. Involvement of adrenal glands in the development of metabolic disorders. The adrenal gland is responsible for the production of hormones that play an essential role not only in reaction to stress (glucocorticoids and adrenalin) but also in the development of high blood pressure (aldosterone), obesity and insulin resistance (glucocorticoids), thus associated with stress‐related metabolic dysfunctions. There is also evidence that chronic stress and sleep disturbance are both associated with hyperactivity of the adrenal gland, the resultant being increased glucocorticoid secretion inducing food intake and weight increase which in turn leads to insulin and leptin resistance. These observations all highlight the relevance of exploring and understanding how the adrenal gland, being the foremost source of steroid production, may be involved in the overall homeostasis.