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Ectopic and Abnormal Hormone Receptors in Adrenal Cushing’s Syndrome¹

Division of Endocrinology, Department of Medicine, Research Center, Hôtel-Dieu du Centre Hospitalier de l’Université de Montréal (CHUM), Montréal, Québec, Canada H2W 1T8

	Abstract

Top Abstract I. Introduction II. Hormonal Regulation of... III. Primary Adrenal... IV. Initial in Vitro... V. In Vivo Demonstration... VI. Investigation Strategy VII. Molecular Mechanisms of... VIII. Ectopic/Abnormal Hormone... IX. An Opportunity for... X. Summary and Conclusions References

 
The mechanism by which cortisol is produced in adrenal Cushing’s syndrome, when ACTH is suppressed, was previously unknown and was referred to as being "autonomous." More recently, several investigators have shown that some cortisol and other steroid-producing adrenal tumors or hyperplasias are under the control of ectopic (or aberrant, illicit, inappropriate) membrane hormone receptors. These include ectopic receptors for gastric inhibitory polypeptide (GIP), ß-adrenergic agonists, or LH/hCG; a similar outcome can result from altered activity of eutopic receptors, such as those for vasopressin (V1-AVPR), serotonin (5-HT₄), or possibly leptin. The presence of aberrant receptors places adrenal cells under stimulation by a trophic factor not negatively regulated by glucocorticoids, leading to increased steroidogenesis and possibly to the proliferative phenotype. The molecular mechanisms responsible for the abnormal expression and function of membrane hormone receptors are still largely unknown. Identification of the presence of these illicit receptors can eventually lead to new pharmacological therapies as alternatives to adrenalectomy, now demonstrated by the long-term control of ectopic ß-AR- and LH/hCGR-dependent Cushing’s syndrome by propanolol and leuprolide acetate. Further studies will potentially identify a larger diversity of hormone receptors capable of coupling to G proteins, adenylyl cyclase, and steroidogenesis in functional adrenal tumors and probably in other endocrine and nonendocrine tumors.

I. Introduction

II. Hormonal Regulation of the Normal Adrenal Cortex

III. Primary Adrenal Cushing’s Syndrome (CS)

IV. Initial in Vitro Evidence of Ectopic Adrenal Membrane Hormone Receptors

V. In Vivo Demonstration of the Functionality of Ectopic or Abnormal Membrane Hormone Receptors

A. Food- and GIP-dependent CS

B. Vasopressin-responsive CS

C. Catecholamine-dependent CS

D. LH-dependent CS

E. LH-dependent adrenal androgen-secreting tumors

F. Serotonin-responsive CS

G. Steroid-responsive CS

H. Other abnormal hormone responses in adrenal CS

VI. Investigation Strategy

A. Initial clinical screening protocol

B. Further characterization of abnormal hormone receptors

C. Systematic clinical screening for ectopic/abnormal hormone receptors

VII. Molecular Mechanisms of Ectopic/Abnormal Hormone Receptors

A. Tissue-specific expression and regulation of membrane hormone receptors

B. Potential mechanisms of ectopic or abnormal hormone receptors

C. Role of ectopic hormone receptors in adrenocortical cell proliferation

VIII. Ectopic/Abnormal Hormone Membrane Receptors in Nonadrenocortical Tumors

IX. An Opportunity for New Pharmacological Therapeutic Strategies

X. Summary and Conclusions

	I. Introduction

Top Abstract I. Introduction II. Hormonal Regulation of... III. Primary Adrenal... IV. Initial in Vitro... V. In Vivo Demonstration... VI. Investigation Strategy VII. Molecular Mechanisms of... VIII. Ectopic/Abnormal Hormone... IX. An Opportunity for... X. Summary and Conclusions References

 
ENDOGENOUS Cushing’s syndrome (CS) is characterized by clinical symptoms and signs resulting from chronic exposure to increased secretion of glucocorticoids (GCs) and other steroids by the adrenal cortex (1, 2, 3). Most frequently, endogenous CS is ACTH dependent, arising from excess ACTH production by pituitary corticotrope adenoma (Cushing’s disease) or from an extrapituitary tumor secreting POMC and ACTH (ectopic ACTH syndrome); rarely, a CRH-secreting tumor causes excessive ACTH production from the pituitary (ectopic CRH syndrome). Less frequently, CS is ACTH independent, as it results from excess secretion of cortisol by benign and malignant adrenocortical tumors or hyperplasias (1, 2, 3, 4). Rare cases of ectopic cortisol production from ovarian tumors that led to ACTH-independent CS have been described (5). Lastly, cortisol hypersensitivity with variable increases in GC receptor numbers has been proposed to explain the clinical features of CS in two patients with low or dysregulated cortisol and ACTH levels and no exposure to exogenous GC (6, 7).

The mechanisms by which cortisol is produced in adrenal CS, when ACTH is suppressed, were previously unknown and referred to as being "autonomous." Studies by several groups have now shown that some of the cortisol-producing adrenal tumors or hyperplasias may actually be under the control of ectopic (or aberrant, illicit, inappropriate) hormone membrane receptors (8, 9, 10). After a brief overview of the regulation of normal adrenocortical function by its main trophic hormones and of the etiologies of adrenal CS, the present review will focus on in vitro and in vivo findings, identifying abnormalities of expression or function of receptors for various hormones in primary adrenal CS. The mechanisms regulating tissue-specific expression of eutopic membrane receptors in the normal adrenal cortex and the potential molecular alterations leading to the ectopic expression of hormone receptors in adrenocortical tumors and hyperplasias will also be discussed. The identification of abnormal membrane hormone receptors in adrenal CS has now opened the field of new therapeutic strategies to control hypercortisolism by interfering with ligand binding to these receptors and will also be presented.

	II. Hormonal Regulation of the Normal Adrenal Cortex

Top Abstract I. Introduction II. Hormonal Regulation of... III. Primary Adrenal... IV. Initial in Vitro... V. In Vivo Demonstration... VI. Investigation Strategy VII. Molecular Mechanisms of... VIII. Ectopic/Abnormal Hormone... IX. An Opportunity for... X. Summary and Conclusions References

 
The normal regulation of adrenocortical function has been the subject of recent reviews (11, 12) and, hence, will be discussed only briefly here. An important site of regulation of the hypothalamic-pituitary-adrenal axis (HPA) is located in neurons of the medial parvocellular part of the hypothalamic paraventricular nucleus (PVN) where CRH and arginine vasopressin (AVP) are produced and travel along their axons to the median eminence to be released in the hypophyseal portal blood system (13, 14). The binding of CRH or AVP to its respective specific receptors CRH-R1 (15) and AVP V3R (16) on corticotrophs of the anterior lobe stimulates the synthesis and maturation of POMC, leading to ACTH secretion (17). Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP), which are also produced in hypothalamic neurons, enhance CRH and ACTH release (18, 19). CRH secretion can be stimulated in the PVN by ₁-adrenoreceptor agonists, serotonin (5-HT_1A) receptor agonists, muscarinic and nicotinic receptor agonists of acetylcholine, histamine, and -aminobutyric acid (GABA_A)_, whereas it is inhibited by GABA_B agonists (14). CRH release is also stimulated by angiotensin II (Ang-II), neuropeptide Y (NPY), cholecystokinin (CCK), and gastrin-releasing peptide, or suppressed by atrial natriuretic peptide (ANP), substance P, somatostatin, and nitric oxide (NO) (14). Several cytokines, including interleukin-1 (IL-1), tumor necrosis factor (TNF-), and IL-6, stimulate CRH, possibly through the production of prostaglandins in brain vascular endothelium (20). ACTH secretion can also be modulated by paracrine/autocrine interactions, as corticotroph cells have been shown to express CRH, which can effectively stimulate ACTH release (21).

ACTH binds to its G protein-coupled membrane melanocortin type 2 receptor (22, 23) to elicit short-term (acute) and long-term (chronic) specific responses, as illustrated in Fig. 1 (24, 25). Activation of the adenylyl cyclase (AC)/cAMP/cAMP-dependent protein kinase (PKA) pathway leads to the phosphorylation of proteins that regulate the early and late steps of steroidogenesis (26, 27). ACTH rapidly (within a few minutes) promotes the mobilization and transfer of free cholesterol to the inner mitochondrial membrane (27). Cloning of the steroidogenic acute regulatory (StAR) protein (28), the subsequent finding of mutations in the StAR gene responsible for the steroid deficiency disease, lipoid adrenal congenital hyperplasia (29, 30), as well as the knockout of this gene in the mouse (31) have identified this ACTH-inducible protein as a key modulator of cholesterol transport into mitochondria. A second protein involved in this process is the peripheral-type benzodiazepine receptor (PBR), which completes the final step of cholesterol delivery to CYP11A1 (P450_scc) for transformation into pregnenolone (32, 33). ACTH also up-regulates the immediate early genes c-fos and c-jun via the PKA pathway (25, 34, 35). A positive feedback loop for the long-term effects of ACTH is established by the hormone up-regulating its own receptor (36, 37).

View larger version (54K): [in this window] [in a new window]   Figure 1. Regulation of steroidogenesis by ectopic hormone receptors in fasciculata cells of adrenal CS. ACTH is the physiological modulator of steroidogenesis in the adrenal cortex. Binding to its receptor (ACTHR) activates AC and leads to cAMP production with cAMP-dependent protein kinase (PKA) activation and phosphorylation of specific TFs (SF-1, NGFI-B, Sp-1, Pbx-1, CREB) that regulate free cholesterol availability and steroidogenic enzymes expression. ACTH also regulates the early steps of steroid synthesis by the direct activation of CYP enzymes. The ectopic expression of membrane hormone receptors functionally coupled to steroidogenesis confers inappropriate sensitivity to adrenocortical cells either to GIP, to catecholamines (E, NE), or to other hormones (LH/hCG, TSH, etc...). These ectopic or abnormal receptors probably regulate steroidogenesis in adrenal CS by mimicking the cellular events triggered by ACTHR activation. N, Nucleus; M, mitochondria; E, epinephrine; NE, norepinephrine. [Modified with permission from N. N'Diaye et al.: Horm Metab Res 30: 440–446, 1998 (10 ). © Georg Thieme Verlag.]

Many ACTH effects are mediated by specific transcription factors (TFs), including orphan nuclear receptors such as nur77 (also called NGFI-B) (39) or steroidogenic factor 1 (SF-1) (40, 41). Indeed, stressful stimuli induce SF-1 and nur77 transcription in corticotrophs and in the adrenal cortex (39, 42). Nur77 and SF-1 both modulate the expression of steroidogenic enzyme genes in the adrenal cortex, nur77 being activated by dephosphorylation and SF-1 by putative PKA-dependent phosphorylation (41, 43, 44).

As an example, SF-1 is involved in the regulation of CYP YP11A (45, 46, 47, 48) and CYP17 (49, 50), where it has been postulated to play a role in constitutive and cAMP-regulated expression. The analysis of the promoter regions of these genes has led to the identification of cAMP-responsive sequences (CRS) and TFs that bind them or synergize cAMP-dependent transcription; general TFs, as cAMP response element (CRE)-binding (CREB) protein and the homeodomain protein Pbx1, both bind CRS and drive cAMP-dependent expression of steroidogenic genes (51, 52, 53, 54, 55). Another ubiquitous TF, Sp1, was shown to regulate basal and cAMP-dependent expression of the CYP11A gene (56). Recent data have suggested that SF-1 is able to mediate cAMP-induced transcription of the CYP17 gene: the proximal CRS (CRS2: –80 to –40) has been identified as a SF-1 binding site (57); moreover, a dominant negative mutation preventing SF-1 binding suppresses cAMP-regulated expression of a reporter gene (58). The coactivator CREB-binding protein (CBP/p300) has been proposed to integrate the effects of TFs such as SF-1, Sp1, CREB, and probably Pbx1 for the regulation of CYP11A and CYP17 genes (59, 60). Moreover, nur77 and nurr1 (nur-related factor 1) positively regulate POMC expression in the pituitary (61, 62). SF-1 up-regulates StAR expression and activity (63). Knockout nur77–/– mice demonstrate no remarkable phenotype (64), suggesting that other members of the nur family play redundant roles, perhaps in humans as well. In contrast, SF-1 appears to be essential for the development and survival of steroidogenic organs, as SF-1–/– mice lack adrenal glands and gonads and exhibit male-to-female sex reversal of their genitalia (65, 66).

Increasing evidence indicates that adrenocortical steroidogenesis is modulated not solely by ACTH but also by multiple circulating and local peptide hormones, neuropeptides, neurotransmitters, ions, and cytokines (11, 67, 68, 69, 70). Both in vivo and in vitro studies have clearly demonstrated that AVP stimulates aldosterone and cortisol secretion in bovine adrenals (71, 72); in rat cells, AVP stimulates aldosterone but not corticosterone secretion (73, 74). However, it stimulates aldosterone (250%) and cortisol (60–260%) secretion from normal human adrenals in vitro (75, 76, 77) via activation of V1-AVP receptors (V1-AVPR) localized mainly in compact cells of the zona reticularis and, to a lesser extent, in the zona glomerulosa (ZG) and fasciculata (68, 74, 78, 79). V2-AVPR were not detected initially in human adrenal cortex tissues (68), but were identified recently by RT-PCR studies (79); their stimulation by DDAVP does not modulate steroidogenesis (79, 80). V3-AVPR (or V1bR) are not detected in the normal human adrenal cortex (79), but are expressed in rat and human chromaffin cells (68, 77, 81), where AVP can stimulate catecholamine release from the adrenal medulla. Thus, AVP could exert significant direct effects on adrenal cortex function, both in endocrine and paracrine modes, but its physiological role has not yet been clearly established. However, in patients with congenital central diabetes insipidus, there is no evidence for clinically significant decreased cortisol secretion (82, 83).

Catecholamines have also been shown to stimulate cortisol and aldosterone secretion in vitro in bovine, pig, and fowl via ß1-adrenoreceptors (11, 84, 85), but this does not appear to occur in human adrenocortical cells (86). Serotonin (5-HT) is another neurotransmitter that may play a role in the control of steroidogenesis (87). 5-HT is able to directly trigger cortisol and aldosterone release, as demonstrated in vitro, in rat, frog, and human adrenal cells (87, 88, 89) but also, indirectly, by stimulating adrenal blood flow (90). The receptor subtype involved in these adrenal effects is still controversial in the rat, but was determined to be 5-HT4 receptor (5-HT₄R) in frogs and humans (88, 89). The 5-HT₄R is positively coupled to the cAMP and calcium pathways. In vivo, 5-HT4 agonists such as cisapride or zacopride induce an increase in aldosterone but not in cortisol secretion in humans (91, 92). Possible paracrine control of steroidogenesis by 5-HT can be proposed since its presence has been demonstrated in human perivascular mast cells and in chromaffin cells of the frog, rat, and mouse adrenals (93, 94, 95). Central 5-HT is known to enhance ACTH release from the pituitary and to activate the systemic renin-angiotensin system (RAS) to stimulate aldosterone secretion. However, no study has established whether these secretory responses can occur within the adrenal gland in vivo.

VIP and PACAP have been shown to play a paracrine role in the secretory activity of the adrenal cortex in the rat, human, and cow, as they are synthesized by adrenomedullary chromaffin cells (18). VIP stimulates aldosterone release from ZG through the activation of selective VIP receptors (VIPR2/VIPR3), whereas it stimulates cortisol secretion moderately through the nonspecific activation of ACTH receptor (ACTHR) (96, 97, 98). VIP/PACAP-induced adrenal steroidogenesis can also be enhanced by an indirect mechanism: indeed, both stimulate catecholamine secretion from adrenal chromaffin cells (99, 100), which in turn elicit a ß-adrenoreceptor-mediated aldosterone release (101, 102). Moreover, cortisol secretion can be raised by increasing the intraadrenal blood flow as it is stimulated by VIP and PACAP (103, 104).

Ang-II, the biologically active peptide of the RAS, and potassium ion are the major regulators of aldosterone synthesis and secretion (2). A decrease in potassium balance activates the RAS, leading to Ang-II, and then to aldosterone release. Ang-II mediates its effect on steroidogenesis via AT1 receptors (AT1R), which are coupled to phospholipases C and A₂ (PLC, PLA₂). It has been demonstrated that Ang-II inhibits the expression of P450c17 at the transcriptional level in ovine adrenocortical cells (105). Moreover, it augments the expression of StAR protein (106). In the rat, Ang-II enhances the transcription of AT1R and P450 aldo synthase (CYP 11B2) in vivo and in vitro (107, 108). However, Ang-II seems to inhibit AT1R expression in bovine and human fasciculata cells (109, 110). The presence of a local RAS in the adrenal cortex suggests that Ang-II can regulate aldosterone production in a paracrine fashion (111) (for review see Refs. 112, 113). Inhibitory signals contribute to maintain aldosterone homeostasis. Dopamine and somatostatin blunt Ang-II-induced aldosterone production (114, 115). The natriuretic peptides ANP and C-type natriuretic peptide (CNP), which are present in the circulation but are also expressed in the adrenal medulla, have been demonstrated to exert an inhibitory action on aldosterone release in vitro (116, 117). ANP also inhibits ACTH and Ang-II-induced cortisol production by decreasing the level of StAR expression (118). Other neuropeptides regulate the steroidogenic function of the adrenal cortex by acting both at the central and adrenal levels, as endothelin 1 (ET-1) (119, 120) and NPY (121, 122) enhance cortisol and aldosterone release.

Recent attention has been drawn to leptin as a negative regulator of the HPA axis. Acute injection of leptin in humans (123) and mice (124) counteracts fasting-induced activation of the HPA axis. This effect is proposed to be driven by a direct action of the peptide, both at the hypothalamic and adrenal levels (125). Leptin and its receptor, Ob-R, are expressed in the pituitary (126, 127) and in human, rat, and mouse adrenal glands (128, 129, 130). Moreover, the adrenal is embedded in adipose tissue, the physiological source of leptin, which acts at the transcriptional level to prevent the stress-induced stimulation of CRH and CYP17 mRNAs in the hypothalamus and adrenal, respectively (131, 132, 133). Other studies have shown opposite effects of leptin on the pituitary where CRH (known to suppress appetite and food intake) and ACTH levels are stimulated, leading to cortisol secretion (134, 135). These discrepancies may arise from anatomic and functional differences in CRH neurons in the PVN where leptin might have inhibitory effects on some and stimulatory effects on other populations of cells. Leptin is induced by GCs (136, 137), resulting in higher plasma levels in CS patients (138, 139).

The integrity of adult adrenal size is maintained by a continuous process of cell division in the ZG and centripetal migration and differentiation into fasciculata cells (140). Chronic stimulation by ACTH induces a phenotypic change of glomerulosa cells into fasciculata cells (141) whereas GCs inhibit this differentiation process namely by reducing P450scc expression (142, 143, 144); it was proposed that GCs may play a role in the functional zonation of the adrenal cortex (11). Indeed, high levels of GC (as high as in the inner adrenal cortex owing to centripetal blood flow) were shown to inhibit the 18-hydroxylation step in ACTH-treated cultures of human fetal adrenals, thus decreasing 18-OH-deoxycorticosterone (DOC) and aldosterone levels (11). In contrast to GC, ACTH can lead in vivo to hypertrophy and hyperplasia of the adrenal cortex, a process that is reversible. Paradoxically, it seems to harbor inhibitory effects on cell proliferation in vitro. A trophic effect is observed after a 2-h exposure to ACTH. This is correlated with a PKA-dependent increase of c-Jun and c-Fos expression (145, 146). After 24 h of stimulation, c-Myc expression is decreased, and inhibition of cell growth is observed (145, 147). Recent data suggest a cAMP-independent proliferation-promoting effect of ACTH (148, 149). Indeed, ACTH was shown to stimulate the mitogen-activated protein (MAP)-kinase pathway in vivo and in vitro, leading to the accumulation of c-Fos, c-Jun, and c-Myc (147, 150). Ang-II is another peptidic hormone that can also activate the MAP-kinase cascade in adrenal cells in a PKC-dependent mechanism (146, 151). In vivo, a chronic stimulation with Ang-II induces ZG hypertrophy. ET-1 also augments cell proliferation in the ZG in vitro and in vivo by interacting with its ET_A receptor, which is specifically expressed in the ZG (119). Chronic treatment with VIP exerts a moderate hyperplasia of ZG in vivo (152, 153). Somatostatin exerts direct antiproliferative effects on the ZG in vivo (115). It can also antagonize the mitogenic action of Ang-II. ACTH stimulates the autocrine production of growth factors (GFs) such as insulin-like growth factor I (IGF-I), IGF-II, and transforming growth factor-ß1 (TGF-ß1), which regulate the trophic and steroidogenic functions of the adrenal cortex in vivo (11, 154). IGF-I and IGF-II have mitogenic effects. IGF-II is more highly expressed in fetal than in adult adrenals (155). In addition, it is highly expressed in hormonally active adrenocortical carcinomas but not in benign tumors, which suggests an important role in tumor acquisition or progression (156, 157). In bovine cells, IGF-I and TGF-ß1 exert opposite effects on adrenocortical function by inhibiting the expression of specific adrenal genes; IGF-I enhances the transcription level of ACTH-R, StAR, and specific steroidogenic enzymes, whereas TGF-ß1 inhibits it (158). TGF-ß1 is thought to play a role in human fetal adrenal remodeling, as it inhibits fetal zone cell proliferation and promotes apoptosis in vitro (159, 160). However, this has not been demonstrated in vivo.

	III. Primary Adrenal Cushing’s Syndrome (CS)

Top Abstract I. Introduction II. Hormonal Regulation of... III. Primary Adrenal... IV. Initial in Vitro... V. In Vivo Demonstration... VI. Investigation Strategy VII. Molecular Mechanisms of... VIII. Ectopic/Abnormal Hormone... IX. An Opportunity for... X. Summary and Conclusions References

 
The incidence of CS has not been determined with great precision. The increasing frequency of subclinical cortisol-secreting adrenal lesions, identified during the evaluation of adrenal incidentalomas, renders precise estimation of the true incidence even more difficult. The incidence of clinical CS secondary to unilateral adrenal adenoma is approximately two cases per million per year (161); this estimate is close to that of 1.7 per million per year for adrenocortical carcinoma, where clinically significant hormonal secretion occurs in 30–60% of cases, including clinical hypercortisolism, in approximately half of the hormonally active cases (162, 163, 164). Since pituitary Cushing’s disease is approximately 3-fold more frequent than primary adrenal disease, its incidence would be close to five to six cases per million per year. When clinically detectable ectopic ACTH secretion is also taken into account, the overall incidence of endogenous CS would reach approximately 10 cases per million per year.

Primary adrenal etiologies account for 15–20% of endogenous CS in adults and are secondary to unilateral tumors in 90–98% of cases (1, 2, 163); in contrast, in prepubertal children, primary adrenal causes are responsible for almost 65% of CS. In adults, some case series have suggested that adenomas and carcinomas are equally responsible for adrenal CS, whereas in other series, adenomas were responsible for up to 80% of cases (165, 166). Cortisol-secreting adrenal carcinomas are 3–4 times more frequent than adrenal adenomas in children. For unclear reasons, adrenal tumors are more frequent in females than in males with a ratio of 4:1 for adenomas and 2:1 for carcinomas (161, 162, 163, 164).

Less than 10% of ACTH-independent CS can be secondary to bilateral adrenal lesions, and their pathophysiology is diverse. Primary pigmented nodular adrenocortical disease (PPNAD) or micronodular adrenal dysplasia can be familial, associated with other tumors such as myxomas, schwannomas, pigmented cutaneous lesions, and peripheral endocrine tumors (Carney’s complex), and linked to unknown genes on chromosome 2 or to mutations of protein kinase A Type 1- located on chromosome 17 (167, 168, 169 169A ). In PPNAD, the overall size of the adrenal gland is usually not enlarged, but is occupied by several small black or brown nodules spread in an otherwise atrophic cortex. High synaptophysin expression in PPNAD nodules suggests a neuroendocrine phenotype of these cells (170). A paradoxical increase in cortisol production is often found in these patients during Liddle’s dexamethasone suppression test (171). In McCune-Albright syndrome, activating mutations of G_s occur in some adrenal cells in a mosaic pattern during early embryogenesis and lead to the formation of adrenal nodules, in which constitutive activation of AC and the steroidogenic cascade produce increased cortisol secretion with ACTH suppression; the internodular adrenal cortex, where the G_s mutation is not present, becomes atrophic (172, 173).

ACTH-independent bilateral macronodular adrenal hyperplasia (AIMAH) is a rare cause of CS, as it is estimated to represent less than 1% of all endogenous cases of this syndrome (1, 2, 3, 4). In a review by Lieberman et al. (174) in 1994, only 24 published cases had been identified, but several other cases and series have been reported since then (175, 176, 177, 178). AIMAH has been described by various terms, including massive macronodular adrenocortical disease (MMAD), autonomous macronodular adrenal hyperplasia (AMAH), ACTH-independent massive bilateral adrenal disease (AIMBAD), and "giant" or "huge" macronodular adrenal disease (175). The clinical syndrome becomes evident during the patient’s fifth or sixth decade and has a relatively even gender distribution when compared with Cushing’s disease or unilateral adrenal tumors, which are more prevalent in women. Most cases have been sporadic, but a few familial cases have been reported as well (179, 180, 181, 182). An activating R201S mutation of G_s was found in the AIMAH tissues of a patient without any other features of McCune-Albright syndrome (183).

	IV. Initial in Vitro Evidence of Ectopic Adrenal Membrane Hormone Receptors

Top Abstract I. Introduction II. Hormonal Regulation of... III. Primary Adrenal... IV. Initial in Vitro... V. In Vivo Demonstration... VI. Investigation Strategy VII. Molecular Mechanisms of... VIII. Ectopic/Abnormal Hormone... IX. An Opportunity for... X. Summary and Conclusions References

 
The concept of ectopic adrenal membrane receptor expression was proposed initially by Robert Ney and his collaborators in 1971 (8, 9, 184). In studying the role of AC in mediating the effects of ACTH in rat adrenal steroidogenesis, only ACTH was capable of stimulating AC in normal cortex membrane preparations; however, in corticosterone-producing rat adrenocortical carcinoma 494, they demonstrated that AC was stimulated by hormones other than ACTH, such as epinephrine, norepinephrine, and TSH (8). Catecholamine effects on AC were induced by ß-, but not by -, adrenergic agonists. Further studies (Table 1) indicated that AC from this tumor was also stimulated by FSH, LH, and slightly by PGE₁) (184), but not by glucagon, insulin, vasopressin, PTH, or calcitonin. Propranolol was able to block the effects of catecholamines but not of other hormones on AC. The illicit hormones exerted no additive or synergistic actions, suggesting that the tumor possessed multiple specific receptors which activated a common AC (Fig. 1). The presence of ectopic and functional ß-adrenergic receptors was also confirmed by other groups (185, 186); high-affinity ß-adrenergic binding sites and AC stimulation were observed in rat adrenocortical carcinoma 494 membranes, but not in normal adrenal membranes (185). A direct effect on steroidogenesis could not be verified in these initial studies, as AC was not efficiently coupled to steroidogenesis in rat adrenal carcinoma 494 (186). The aberrant response of AC to various hormones is not a universal phenomenon, as the AC of the Y1 mouse adrenocortical tumor cell line was found to be stimulated by ACTH, but not by epinephrine, PTH, insulin, glucagon, TSH, or PGE₁ (187). The presence of ectopic - adrenergic receptors stimulating guanylate cyclase and cGMP production was also demonstrated in rat adrenal carcinoma 494 (188, 189).

Other in vitro studies have further supported the functional coupling of several, most frequently G protein-linked, membrane hormone receptors to steroidogenesis in some human adrenocortical benign and malignant tumors (Table 1). Millington et al. (190) investigated the effects of various hormones on the secretion of steroids in a human feminizing adenocarcinoma secreting mostly estrogens and androgens, but also some GC. AC activity was stimulated more by PRL, human placental lactogen, LH, and FSH preparations than by ACTH; insulin inhibited AC slightly, while TSH was without effect. In tumor explant culture, estrone and estradiol secretion was stimulated by PRL, insulin, and ACTH, but little by LH or GH. Androstenedione secretion was augmented by LH, GH, PRL, and ACTH. The synthesis of 11-hydroxycorticosteroids was stimulated by LH, GH, and PRL, but very little by ACTH. It must be stressed that hormone preparations available at that time were not pure and that contamination was quite possible. Matsukura et al. (191) studied AC activity in human cortisol-secreting adrenal tissues from adenomas, adenocarcinoma, and primary nodular hyperplasia (AIMAH), compared with normal adrenals and bilateral hyperplasias from pituitary Cushing’s disease. In normal tissues, only ACTH and PGE₁ stimulated AC activity; in most adenomas, AC activity was increased by norepinephrine, in some by epinephrine, and in a few by TSH, LH, or Ang-II. In a case of AIMAH, AC was stimulated by glucagon and ACTH only. No stimulation of AC was found in adrenal carcinoma tissue. Hirata et al. (192) demonstrated the presence of high-affinity ß-adrenergic binding sites in two of three cortisol-secreting adenomas, but not in the normal adrenal cortex or in one case of aldosterone-producing adenoma; furthermore, epinephrine stimulated cortisol secretion in cultured tumor cells from one of the patients with an adenoma, and Katz et al. (193) studied six human adrenal carcinomas with diversified steroidogenic activities and compared them with the normal adrenal cortex from three individuals; AC was stimulated by ß-adrenergic agonists in four of six tumors but not in normal tissues. In one tumor examined for other hormone responses, AC was also stimulated by TSH, but not by glucagon or hCG. In two cases, membranes from metastatic adrenocortical cancer were compared with the primary tumor and had lost stimulation of AC by epinephrine or ACTH. Specific high-affinity ß-adrenergic binding sites were detected only in tumors in which AC was stimulated by ß-adrenergic agonists. In contrast, Saez et al. (194) did not find any AC responsiveness to norepinephrine, glucagon, and TSH in crude adrenal membranes from 11 patients with adenomas and carcinomas.

The aberrant expression of LH/hCG receptors was also previously reported in vitro in androgen-secreting adrenal adenomas (195, 196). Testosterone production was stimulated by hCG and ACTH in adrenal adenoma cells in culture, while only ACTH but not hCG was able to stimulate secretion of cortisol, testosterone, and other steroids from the adjacent normal adrenal cortex (195); binding studies performed on cell membranes from hCG-responsive adrenal adenoma demonstrated high-affinity (0.14 nM) binding capacity (198 fmol/g). A preliminary report of the presence of LH/hCG receptor in a cortisol-secreting adrenocortical carcinoma was presented recently (197).

Willenberg et al. (198) investigated the adrenal adenoma of a 62-yr-old woman who presented CS with no particular clinical characteristics; striking lymphocytic infiltration of the adenoma was identified at histology. In contrast to normal control human adrenals or other cortisol-secreting adenomas or carcinomas, immunostaining revealed CD45 and CD68-positive macrophage-like cells in this patient’s adenoma, and these cells are a major source of IL-1. Type I IL-1 receptor, which is not a seven-transmembrane G-coupled-receptor, was also found to be aberrantly expressed in the adenoma, by in situ hybridization and RT-PCR, but not in the normal adrenal cortex or other tumors. In cells dispersed from the adenoma, cortisol secretion was stimulated 2.6-fold by IL-1ß, but poorly by ACTH (198); in normal adrenocortical cells or other cortisol-secreting adenomas, cortisol secretion was increased by approximately 1.5-fold during incubation with IL-1ß. Since infiltration of mononuclear cells occurs in 15% of adrenal tumors, it will be of interest to further explore the prevalence of abnormal cytokine receptor expression in adrenal hyperplasias and tumors.

	V. In Vivo Demonstration of the Functionality of Ectopic or Abnormal Membrane Hormone Receptors

Top Abstract I. Introduction II. Hormonal Regulation of... III. Primary Adrenal... IV. Initial in Vitro... V. In Vivo Demonstration... VI. Investigation Strategy VII. Molecular Mechanisms of... VIII. Ectopic/Abnormal Hormone... IX. An Opportunity for... X. Summary and Conclusions References

 
The proposed concept of ectopic hormone receptors had been demonstrated in vitro only, until it found a clinical manifestation of its significance, in vivo, with the description of food-dependent CS (199); this resulted from ectopic adrenal expression of the receptor for a gastrointestinal hormone called gastric inhibitory polypeptide or GIP (200, 201).

A. Food- and GIP-dependent CS
Hamet et al. (199) were the first to identify "food-dependent" cortisol production in a 41-yr-old male patient presenting with CS secondary to a unilateral adrenal adenoma and periodic hormonogenesis. Plasma cortisol was consistently low in the morning or during fasting, but increased to abnormal levels after meals; food-induced elevations of plasma cortisol were not suppressed by high oral doses of dexamethasone. AC activity in the resected adrenal adenoma membrane preparation was stimulated 27% by ACTH and 62% by vasopressin, but not by FSH, glucagon, or Ang-II; the effects of various gastrointestinal hormones were not examined in this case. Another female patient with CS secondary to an adrenal adenoma had been previously reported to have "persistent diurnal cortisol secretory rhythm" (202); the low fasting plasma cortisol levels in the morning increased during the day at the presumed, but not indicated, meal times, suggesting that this patient also had food-dependent CS.

Two patients with bilateral AIMAH and food-dependent cortisol production were studied in detail a few years later and allowed to clarify the pathophysiology of this syndrome (200, 201). The first patient, a 48-yr-old French-Canadian woman, presented with typical symptoms of CS, which had become manifest during the previous 2–3 yr (200). Initial investigation revealed low plasma cortisol levels, fasting in the morning, and higher levels during the day, whereas plasma ACTH was always suppressed. The suspicion that cortisol production was regulated by a gastrointestinal hormone came from the observation that plasma cortisol was stimulated by oral administration of glucose or by lipid-rich or protein-rich meals, but not by intravenous glucose. In addition, somatostatin pretreatment inhibited the cortisol-stimulatory effect of oral glucose. A review of the various secretagogues of gastrointestinal hormones indicated that only GIP and the glucagon-like peptides (GLPs) were stimulated significantly by oral glucose and lipids, and to a lesser extent by proteins. Plasma cortisol levels were correlated with plasma GIP concentrations during the various test meals. In vivo GIP infusion, to reproduce physiological postprandial concentrations, augmented cortisol production in the patient, but not in four normal controls. In the patient, plasma cortisol was stimulated by the administration of ACTH but not by CRH, glucagon, insulin-induced hypoglycemia, pentagastrin, or AVP. The presence of GIP receptors (GIPRs) in adrenal tissues was supported by adrenal imaging after the injection of [¹²³I]-GIP in vivo (200). The incubation of dispersed adrenal cells in vitro confirmed GIP-mediated cortisol secretion in the patient’s cells, whereas no cortisol response to GIP was found in normal adult or fetal adrenal cells or in other cortisol- or aldosterone-secreting adenomas (200); there was no stimulation of cortisol production in the patient’s adrenal cells after in vitro incubation with secretin, CCK, VIP, substance P, bombesin, calcitonin gene-related peptide, glucagon, vasopressin, ANP, CRH, TRH, GHRH, neurotensin, or neurokinin A. It was thus concluded that food-dependent cortisol secretion resulted from the abnormal responsiveness of adrenal cells to the physiological secretion of GIP; "illicit" or ectopic GIPR expression on adrenal cells (Figs. 1 and 2) presumably were the basis for this new etiology of CS (200).

View larger version (45K): [in this window] [in a new window]   Figure 2. HPA axis in GIP-dependent CS. The ectopic adrenal expression of the GIPR (in red) has been identified both in bilateral macronodular adrenal hyperplasia (left of figure) or in unilateral adrenal adenomas (right side of figure). After food ingestion, GIP is released in physiological concentrations by K cells from the duodenum and small intestine and binds to the ectopic adrenal GIPR; this results in postprandial supraphysiological increases of plasma cortisol (full black lines) which exerts its negative feedback on CRH and ACTH synthesis. In the absence of food ingestion (low plasma GIP levels), the suppressed levels of plasma ACTH (dashed blue lines) leads to decreased occupation of the ACTHR (decreased expression) and low fasting levels of plasma cortisol. A somatic postzygotic mutation occurring in a single cell leading to GIPR expression would eventually result in the growth of a GIP-dependent monoclonal unilateral cortisol-secreting adenoma with adjacent and contralateral adrenal cortex atrophy (right side of figure). A somatic mutation occurring during early embryonal life and responsible for ectopic GIPR expression in the progenitor cells of the adrenal cortex (polyclonal) would be responsible for the long-term development of nonfamilial GIP-dependent bilateral macronodular adrenal hyperplasia and CS (left hand side of figure).

Food- or GIP-dependent CS has now been identified in 13 patients with AIMAH (139, 200, 201, 205, 206, 207, 208) and in seven with unilateral adenoma (199, 205 208A, 213), as summarized in Table 2. At pathological examination, no distinctive features were reported, compared with non-GIP-dependent cortisol-secreting adenomas or bilateral macronodular hyperplasia, except in one case (207). This patient was described in a preliminary report to have facial pigmented spots, a blue nevus on one leg, lipofuscin pigments in bilateral adrenal macronodules, and a periadrenal schwannoma suggestive of Carney’s complex without any family history; a full description has not yet been published, but in vitro studies clearly confirmed GIP-induced stimulation of cortisol secretion by adrenal cells (205). In two cases of AIMAH, the patient initially presented with a unilateral lesion and developed contralateral enlargement only later in time (206 208A ). Except for three patients [the first patient described with food-dependent CS but not proven to be GIP-dependent (199) and two recent ones with AIMAH (GIPR overexpression not yet confirmed)], all other patients are females; adrenal CS is more frequent in females (161), but it remains to be seen whether an even higher female frequency will be found in GIP-dependent CS and what molecular mechanism underlies this sex distribution. Average age at the time of diagnosis may be somewhat greater in patients with AIMAH than in patients with unilateral adrenal adenoma (Table 2) (174, 175); the youngest patient with a unilateral adenoma was only 15 yr old. In GIP-dependent CS, chronic GIP-induced hypercortisolism eventually leads to suppression of CRH and ACTH; this suppression, coupled with low GIP levels in the fasting state, is responsible for the decreased plasma cortisol levels, which can be accompanied by symptoms of relative cortisol insufficiency (201, 209). However, in certain patients (Table 2), fasting plasma cortisol levels were not particularly low, indicating that GIP-dependent CS should not be excluded without performing a test meal (139, 206); this finding could indicate that subpopulations of adrenal cells in the tumor or hyperplasia have lost their GIP dependency and are secreting cortisol under different mechanisms, or that more than one abnormal receptor regulating cortisol production are expressed in these cells. In one patient with food-dependent AIMAH but in whom fasting plasma cortisol was relatively elevated, Pralong et al. (139) reported that, in addition to GIP, leptin also aberrantly stimulated cortisol secretion in dispersed adrenal cells; thus, the potential presence of more than one abnormal receptor may modify the phenotypic appearance. The potential presence of ectopic GLP-1 receptors has been excluded to date by the lack of stimulation of cortisol production after GLP-1 administration, either in vivo or in vitro (139, 206, 210). In one patient with GIP-dependent AIMAH, plasma ACTH and cortisol responses to CRH were still preserved, presumably because the intermittent food-dependent stimulation of cortisol had not yet completely suppressed the HPA axis (208). In a female patient with hirsutism and a unilateral adenoma, both adrenal androgens and cortisol were found to be stimulated by food intake in vivo and GIP in vitro (213); hypercortisolism was modest and ACTH was not fully suppressed.

De Herder et al. (209) used in situ hybribization to demonstrate abundant GIPR mRNA in adrenal adenoma cells from their patient with GIP-dependent CS; this signal was not present in the adenoma from a patient with non-food-dependent CS, but was not examined in the normal adrenal cortex in this initial study. Using RT-PCR amplification, N'Diaye et al. (219) demonstrated pronounced adrenal GIPR overexpression in adrenal adenoma or hyperplastic tissues from GIP-dependent CS compared with the normal human pancreas, normal adult or fetal adrenal cortex, or non-GIP-dependent adrenal CS tissues. A small amount of GIPR mRNA was detected in normal fetal and adult adrenal tissues after at least 35 cycles of amplification and hybridization with the labeled cDNA but was not coupled efficiently to steroidogenesis. Sequence analysis of the full-length cDNA of normal and GIP-dependent adrenal tissues revealed no mutation of GIPR in the affected adrenal tissues (219); similar proportions of isoforms lacking exons 4 and 9 were identified in normal and GIP-dependent adrenals. Chabre et al. (210) confirmed the presence of the same overexpressed GIPR isoforms in a GIP-dependent adenoma by RT-PCR and sequencing; no GIPR bands could be detected in the atrophic adrenal cortex adjacent to the tumor or in normal adult adrenals, but only ethidium bromide staining was used. The ACTHR was found to be expressed at a lower level in GIP-dependent adenoma compared with normal tissues (210); this may be secondary to the chronic suppression of endogenous ACTH, which is known to up-regulate ACTHR expression (36, 37). If the relative suppression of ACTHR in GIP-dependent adrenal tissues is confirmed in further studies, this would indicate that GIP cannot substitute for ACTH in inducing the expression of ACTHR; it must be noted, however, that plasma GIP levels are only elevated transiently postprandially, which is different from conditions where ACTH is elevated chronically. GIPR overexpression was confirmed in other cases (Table 2) of GIP-dependent adrenal macronodular hyperplasias (205, 206, 208 208A ) and adenomas (205 208A, 210, 213) and was not demonstrated in non-GIP-dependent CS adrenal tissues (205, 210, 213, 219) or the human adrenocortical carcinoma cell line H295 (211). GIPR overexpression was detected, even in the early stages of adrenal hyperplasia (206). The small amount of GIPR mRNA sometimes found in normal fetal or adult adrenal tissues after amplification was not efficiently coupled to steroidogenesis (219) and may reflect a low number of GIPR in endothelial cells (214) rather than in adrenocortical cells. Thus, the concept of functional ectopic receptors remains valid in explaining the pathophysiology of GIP-dependent CS (Figs. 1 and 2).

It has been reported that the in vitro cortisol-stimulating effects of GIP are coupled to an increase of cAMP, but not of IP₃ production (205, 210). In studying GIP-dependent adrenal cells in primary culture, GIPR down-regulation by its own ligand has been demonstrated, as assessed by the induction of steroidogenic enzyme expression, cortisol secretion, or GIPR mRNA levels by in situ hybridization and RT-PCR studies (205, 220). By stimulating steroidogenic enzyme activity, ACTH pretreatment of cells increased the GIP-induced cortisol response but did not appear to modify GIPR expression directly (205).

Stimulation of thymidine incorporation into newly synthesized DNA by GIP was observed in primary cultures of adrenal cells from GIP-dependent CS, but not in normal cells (210). Activation of p42-p44 MAP kinases was observed after treatment of pathological cells with GIP (210). Depending on the cell culture conditions used, ACTH can be shown to inhibit or stimulate markers of cell proliferation in adrenal cells. In the studies by Lebrethon et al. (205), under conditions where ACTH inhibited thymidine incorporation in normal and GIP-dependent adrenal cells, GIP was also found to suppress DNA synthesis only in GIP-dependent, and not in normal adrenal cells. Such results suggest that GIP is possibly capable of regulating cell proliferation, in addition to steroidogenesis, in these tissues; however, cell growth stimulation by GIP has not yet been clearly demonstrated.

It should be stressed that food-induced cortisol secretion has been reported in some non-GIP-dependent CS. Bercovici et al. (221) described a patient with pituitary Cushing’s disease in whom ACTH and cortisol were increased strikingly after mixed meals. ACTH secretion was stimulated by protein-rich meals, but not by oral glucose or lipid-rich meals. Intravenous infusion of amino acids was capable of inducing this response, while octreotide administration did not modify urinary cortisol levels. It was concluded that the pituitary corticotroph adenoma of this patient retained the capacity that normal corticotroph cells have to enhance their release of ACTH after protein ingestion. It has been shown very clearly that, in normal individuals, mixed meals produce an increase in ACTH release and in plasma cortisol levels; this is more evident at lunchtime than after breakfast, when the diurnal peak of ACTH and cortisol may mask the response (222, 223, 224). This stimulation is of hypothalamic-pituitary origin and is abolished by dexamethasone administration (225). It is believed that the effect may be secondary to the heightened serotonin production and related to tryptophan content in the meal (224). -Adrenergic agonists can also increase postprandial stimulation of ACTH (226).

B. Vasopressin-responsive CS
A large proportion of pituitary corticotroph adenomas have been shown to augment their ACTH release after LVP administration, resulting in increased plasma cortisol levels (227, 228). In contrast, in adrenal CS, where ACTH is suppressed, it is expected that plasma cortisol should not increase after LVP administration (229). However, abnormal adrenal stimulation of cortisol secretion in response to exogenous AVP or LVP administration has been described in canine (230) and human ACTH-independent CS, secondary to unilateral adrenal adenomas, carcinomas, or AIMAH (Table 3).

Horiba et al. (234) reported two male Japanese patients with bilateral macronodular adrenal hyperplasia and clinical CS in whom im injection of 10 IU LVP increased plasma cortisol 2.3- to 2.6-fold, while plasma ACTH remained undetectable; there were no ACTH or cortisol responses to CRH or dexamethasone. Upon pathological examination, the glands were replaced by macronodules composed of compact and clear cells, but there were some regions of cortical internodular atrophy. In dispersed adrenal cells from both patients, LVP stimulated cortisol secretion (2.8- to 3.2-fold) more efficiently than ACTH. In seven other patients with CS and unilateral adenoma, LVP injection resulted in small increases of plasma cortisol, varying between 9.8 and 25.3%. In four normal subjects pretreated with 2 mg dexamethasone at bedtime and 0.5 mg on the morning of the test, LVP injection elevated plasma cortisol 1.6- to 1.8-fold (up to 45 nmol/liter from basal levels of 20.9 nmol/liter). An exaggerated 2.6-fold rise in plasma cortisol after 10 IU of LVP was also reported in a patient with a unilateral cortisol-secreting adenoma and mild ACTH-independent CS (235). Intracellular calcium flux in dispersed tumor cells was stimulated by AVP and inhibited by a V1-AVPR antagonist. Using RT-PCR amplification, the V1-AVPR signal was stronger in the cortisol-secreting tumor than in the normal gland; there was a faint V2-AVPR signal in normal and tumoral adrenal tissues, and no V3-AVPR in either.

A 36-yr-old female American patient with CS and AIMAH presented an unusual association with orthostatic hypotension (80). Exogenous AVP, but not desmopressin, triggered large elevations of plasma cortisol (3.4-fold) and aldosterone (67-fold) levels. During upright posture and hypotension, cortisol and aldosterone secretion increased, despite the suppression of ACTH and renin levels. AVP, which normally rises during upright posture and even further in orthostatic hypotension, remained below the limit of assay detection, until the correction of hypercortisolism. Under dexamethasone suppression, plasma cortisol, aldosterone, and androgens were elevated by exogenous AVP in the patient, but not in the controls. Cells freshly dispersed from the diffuse adrenal hyperplasia displayed higher cortisol stimulation (4.2-fold) during incubation with AVP than normal adrenal cells (1.3-fold); the cortisol response was mediated by V1-AVPR, as shown by the effects of V1 antagonists and the lack of effect of V2 agonists. The presence of V1-AVPR was supported by binding studies, intracellular Ca²⁺ flux studies, and RT-PCR amplification of mRNA for all three AVPR. The binding studies revealed a similar V1-AVPR affinity (2.63 nM) in AIMAH adrenal cells, compared with membranes from human glomerulosa-rich normal adrenal cells or myometrium (236). The ED₅₀ of AVP on [Ca²⁺]_i was similar in the adrenal cells of the patient (0.9 nM) compared with glomerulosa-rich cells (1.4 nM) from normal adrenals (76). Interestingly, CRH administration stimulated cortisol in vivo but not in vitro without any stimulation of ACTH; it is possible that CRH increased the adrenal production of vasopressin (68) and cortisol in a paracrine manner. Alteration of the V1-receptor-effector system was not limited to the adrenal tissues of this patient, as there was also an abnormal, prolonged vascular vasoconstrictive response to AVP, compared with the arterioles of normal or hypertensive subjects. The persistence of decreased stimulation of plasma vasopressin and endothelin levels during postural hypotension, several months after correction of the hypercortisolism, also raised the possibility of an exaggerated V1-AVPR signal at the hypothalamic level in this patient. The causal relationship between abnormal V1-AVPR-mediated-responses and postural hypotension remains uncertain (80). Another male Japanese patient with AIMAH and CS was found to have a 1.8-fold increase in plasma cortisol after LVP injection (237); food intake, GIP infusion, octreotide, and CRH were without effects. Removal of the large bilateral macronodular adrenals showed no areas of internodular atrophy; LVP stimulated cortisol production in cells freshly dispersed from a macronodule. Stimulation of plasma cortisol by administration of 0.2 IU AVP was noted in a Japanese man with AIMAH and coincident multiple adenomatous polyps and colon cancer (238); a point mutation of the APC gene was revealed in the colon cancer but not in the adrenal nodules.

In a retrospective study of 26 patients with CS secondary to unilateral cortisol-secreting tumors, Arnaldi et al. (79) observed an increase of plasma cortisol greater than 30 ng/ml after LVP testing in 27% of cases (five adenomas and two carcinomas). Quantitative RT-PCR assay of V1-AVPR showed that the levels of message were similar in 20 cortisol-secreting adenomas, compared with three normal adult adrenals; the levels were lower in 19 adrenocortical carcinomas, but there was a large overlap with adrenal adenomas. The normal adrenal glands and the majority of tumors also expressed low amounts of V2-AVPR, but no V3-AVPR. Only six of the patients for whom adrenal tumor material was available had undergone LVP testing; responders had somewhat higher V1-AVPR concentrations in their tumors than nonresponders, but the levels were not higher than in normal adrenal tissues. In one patient with an in vivo cortisol response (1.6-fold) to LVP, the AVP-induced cortisol secretion (2-fold) of perifused adrenal cells was inhibited by V1-AVPR antagonists.

The demonstration of an exaggerated cortisol response to pharmacological levels of exogenous vasopressin does not constitute direct evidence that fluctuations of endogenous AVP levels are the main regulator of steroidogenesis in these patients. This was illustrated in a male patient with AIMAH who was shown to have increased plasma cortisol in response to upright posture and administration of 10 IU AVP (86); however, the modulation of endogenous AVP levels by water dilution or hypertonic saline infusion did not modify plasma cortisol levels. In addition, in vivo administration of a V1-AVPR antagonist inhibited the response of cortisol to exogenous AVP, but not to upright posture. In fact, this patient was found to have ectopic ß-adrenergic receptors (see Section V.C.) in his adrenal tissues; it is believed that pharmacological AVP levels stimulated catecholamine release, including from the adrenal medulla (68), and then mediated cortisol release in this case. Further support comes from the fact that there was no evidence of V1-AVPR in his adrenal tissues (N. N’Diaye and A. Lacroix, unpublished observation).

Daidoh et al. (239) studied a 49-yr-old man with very large bilateral AIMAH and severe CS; intravenous injection of small amounts of AVP (0.3 IU) increased plasma cortisol 3.7-fold without any detectable rise in ACTH. Similarly, insulin-induced hypoglycemia elevated plasma AVP and cortisol without any increase in plasma ACTH; catecholamine effects were not studied however. Upright posture augmented plasma AVP and cortisol. Oral administration of the V1-AVPR antagonist OPC-21268 for 8 days decreased urinary free cortisol levels, but potential spontaneous fluctuations of cortisol secretion were not evaluated for long periods. It was further shown, in dispersed adrenal cells, that AVP stimulated cortisol secretion in AIMAH cells but not in normal control cells, and that this effect was inhibited by OPC-21268; GIP was without effects on AIMAH cells, but catecholamine and insulin were not tested directly. We recently studied a 50-yr-old American woman with CS and AIMAH in whom plasma cortisol was stimulated by upright posture (1.7-fold) and exogenous AVP (3.4-fold), but not by dDVAP (240). In this patient, we were able to demonstrate that plasma cortisol was inhibited by water loading (24% decrease), and elevated during hypertonic saline infusion (1.7-fold). This patient was also found to have abnormal responses to ß-adrenergic receptor agonists (see Section V.C.), in addition to the abnormal V1-AVPR response in her adrenals. These last two cases represent the first demonstrations of fluctuations in plasma cortisol levels in parallel with small physiological changes in endogenous vasopressin levels. All the previously reported cases of cortisol stimulation by lysine- (231, 232, 233, 234, 235, 237) or arginine-vasopressin (80) were related to exogenous pharmacological amounts. In these last two patients, as in another patient (80), plasma vasopressin was found to be suppressed to undetectable levels basally and showed only a very modest increase upon potent physiological stimulation. This may be due to the suppressive effects of hypercortisolism on vasopressin gene expression (241). It has also been postulated that abnormal V1-AVPR may modify vasopressin production via a short loop regulation mechanism in hypothalamic nuclei (80).

An abnormal increase of plasma cortisol in response to vasopressin administration was also noted in patients with preclinical bilateral macronodular adrenal hyperplasia (242). Recently, an exaggerated plasma cortisol response to LVP was seen in a 67-yr-old woman with CS and bilateral macronodular adrenal hyperplasia, whose brother had died after bilateral adrenalectomy for CS and AIMAH (182); the precise nature of the abnormal hormone receptor implicated is unknown, but this constitutes the first demonstration of abnormal hormone responsiveness in familial AIMAH.

Since V1-AVPR are present in the normal adrenal cortex and modulate modest effects of vasopressin on steroidogenesis, the exaggerated steroidogenic responses to vasopressin in these patients would be secondary to the abnormal function of an "eutopic" receptor-effector system, rather than to the presence of an ectopic receptor. V1-AVPR mRNA levels were found to be expressed either at higher (235) or similar (79, 80) levels, compared with normal control adrenal tissues. The binding affinity and dose response of intracellular calcium flux for V1-AVPR noted in the adrenal tissues of a patient with AIMAH (80) were not different from those reported in other normal tissues. Thus, no evidence of ectopic receptor or gross overexpression of the eutopic V1-AVPR has been presented to date; the molecular mechanisms leading to the abnormal response of V1-AVPR or its effector system, which would increase the response to AVP, remain to be elucidated.

Recently, V3-AVPR were shown to be expressed ectopically in a series of bronchial carcinoids secreting ACTH (243). A large proportion of patients with Cushing’s disease, but not normal individuals, secrete ACTH in response to DDAVP (244, 245). V3-AVPR were found to be overexpressed in corticotroph adenomas (229); as DDAVP can also bind in part to V3-AVPR, this may explain the effects of DDAVP on ACTH release in Cushing’s disease. Thus, stimulation of cortisol levels after vasopressin administration in CS cannot directly distinguish between pituitary corticotroph adenoma, ACTH-independent primary adrenal tumor or hyperplasia, or relatively well differentiated carcinoid tumors producing ACTH.

C. Catecholamine-dependent CS
Catecholamines are known to modulate HPA activity. Activation of _1-adrenoreceptors in the PVN leads to CRH release with increased plasma levels of ACTH and cortisol (14). Administration of ß₁- or ß₂-adrenergic agonists or antagonists has no effect on ACTH or cortisol secretion (246). Peripherally administered _1-adrenoreceptor agonists fail to activate the HPA, as the blood-brain barrier prevents their access to the PVN. Direct adrenal stimulatory or inhibitory effects of catecholamines on GC or mineralocorticoid secretion have been noted in several species, but are limited to aldosterone secretion in humans, where cortisol secretion is unaffected (11).

As discussed in Section IV, the abnormal presence of ß- adrenergic receptors or the activation of AC activity by catecholamines has been reported in vitro in several cases of human adrenal tumors associated with CS (191, 192, 193); no evidence of such receptors has been found in the normal adrenal cortex. However, the clinical expression of this abnormality was appreciated only recently in two patients. A 56-yr-old French-Canadian man with AIMAH and CS (86) was shown to have ACTH-independent overproduction of cortisol and aldosterone during elevations of endogenous catecholamines level (upright posture, insulin-induced hypoglycemia, and EKG stress test). Augmented plasma cortisol during upright posture was decreased after pretreatment with the ß-adrenergic antagonist, propranolol; in contrast, this did not occur after inhibition of the RAS system with captopril or losartan, or of AVP with a V1-AVPR antagonist. Isoproterenol infusion stimulated cortisol (2.1-fold) and aldosterone (2.2-fold) secretion in the patient, but not in normal subjects, in whom ACTH had been suppressed by dexamethasone. Plasma cortisol was not influenced by mixed meals, or administration of TRH, GnRH, glucagon, or cisapride; as discussed previously, a late increase of cortisol after AVP administration was believed to result from stimulation of release of adrenomedullary catecholamines. High-affinity binding sites compatible with ß₁-adrenergic receptor (ß₁-AR) or ß₂-AR were found in the adrenal tissues of the patient, but not in the controls. They were efficiently coupled to steroidogenesis (Fig. 1), as shown by AC stimulation with isoproterenol in vitro and catecholamine-induced steroidogenesis in vivo (86). Further molecular studies are needed to properly characterize the ß-adrenergic receptor subtype expressed in hyperplastic adrenal tissues and to determine whether or not it is mutated.

Another 50-yr-old American woman with CS and AIMAH (240) was found to have abnormal responses to catecholamines in addition to an exaggerated response to AVP (described previously in Section V.B.). In this patient, plasma cortisol had risen after upright posture (1.7-fold) and exogenous AVP (3.4-fold), but also after insulin-induced hypoglycemia (2.7-fold), while ACTH remained suppressed. Infusion of isoproterenol for 30 min increased plasma cortisol from 323 to 630 nmol/liter, which returned rapidly to baseline when the infusion was discontinued. Pretreatment of the patient with the angiotensin receptor type-1 antagonist losartan did not prevent the elevation of plasma cortisol during upright posture. There were no increases of plasma cortisol after mixed meals, GnRH, TRH, glucagon, or cisapride. It was concluded that cortisol secretion was mediated by the abnormal presence and function of ß-adrenergic and V1-AVPR, and medical therapy with the ß-blocker propranolol was proposed to the patient; she did not tolerate this medication well and elected to undergo surgery in her home city (tissues not available).

D. LH-dependent CS
The LH/hCG receptor (LH/hCGR) normally activates AC and PLC to stimulate gonadal steroidogenesis (247). The receptor is mainly expressed in gonadal tissues, but also in other tissues, including the uterus, fallopian tubes, placenta, brain, hypothalamus, and prostate (248); recently, the presence of LH/hCGR was identified in the zona reticularis of the human adrenal (249) by immunohistochemistry and in situ hybridization. hCG stimulates DHEAS secretion in human fetal adrenal cells (250).

A 63-yr-old French-Canadian woman was studied for CS and AIMAH (251). Retrospectively, she described having gained between 18–22 kg during each of four full-term pregnancies, with Cushingoid fat distribution, but without high blood pressure, purple skin striae, or hirsutism. Her weight returned rapidly to baseline after delivery with symptoms of lack of appetite, nausea, and fatigue, which subsided within 2–3 months. Chronic hypercortisolism became clinically manifest only 10 yr after menopause (Fig. 3). Cortisol production was increased by the in vivo administration of GnRH, hCG, and recombinant human LH (hLH). Plasma free testosterone and estradiol were also augmented by hLH administration. Abnormal stimulation of cortisol, free testosterone, and DHEAS production was also evoked in this patient by oral intake of cisapride and metoclopramide, two 5-HT₄R agonists (251). Administration of the long-acting GnRH analog leuprolide acetate initially increased LH and FSH secretion, which was paralleled by a rise in cortisol secretion; however, this was followed within 10 days by suppression of endogenous LH and FSH levels and normalization of cortisol production. Stimulation of cortisol by hCG and recombinant hLH, but not by FSH, suggests that a functional adrenocortical LH/hCGR was coupled to steroidogenesis (Fig. 3); the lack of stimulation by GnRH, when LH levels were suppressed by chronic administration of leuprolide acetate, excludes an adrenal GnRH receptor. Studies of normal adult controls did not indicate any coupling of LH/hCGR to adrenal synthesis of cortisol or DHEAS. Abnormal stimulation of plasma cortisol after GnRH and LH administration was also found in one woman with bilateral macronodular adrenal hyperplasia and normal urinary cortisol levels, which did not suppress normally with dexamethasone (242). This suggests that diverse ectopic hormone receptors can be present in preclinical bilateral macronodular adrenal hyperplasia.

View larger version (22K): [in this window] [in a new window]   Figure 3. HPA axis in CS secondary to the ectopic adrenal expression of LH/hCGR. Adrenal expression of the LH/hCGR in the adrenal cortex is illustrated. Occupation of this receptor either by hCG of placental origin or by LH of pituitary origin induces cortisol secretion, which exerts negative feedback inhibition on CRH and ACTH production (upper panel). The development of bilateral adrenal hyperplasia and hypercortisolism is produced transiently and reversibly due to occupation of the receptor by hCG during pregnancies; delivery is followed by a transient period of hypocortisolism (upper and lower panels). At the time of menopause, a sustained elevation of LH levels follows a decrease in ovarian estrogen production and results in a progressive increase of bilateral adrenal hyperplasia and hypercortisolism. Administration of long-acting leuprolide acetate initially induced transient stimulation of LH and cortisol, followed by long-term suppression of LH and restoration of normal cortisol production.