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Department of Endocrinology (J.B., L.G., X.B.), Centre Hospitalier Universitaire Cochin & Institut Cochin, Institut National de la Santé et de la Recherche Médicale (INSERM) U567, Centre National de la Recherche Scientifique (CNRS) UMR8104, IFR 116, Université Paris V-René Descartes, 75014 Paris, France; European Institute for Peptide Research (Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23) (V.C., E.L., C.D., J.-M.K., H.V., H.L.), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U413, Unité Associée CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France; and Department of Endocrinology (G.B., P.E.), Centre Hospitalier Régional of Orléans, 45067 Orléans, France
Address all correspondence and requests for reprints to: Hervé Lefebvre, Institut Fédératif de Recherche Multidisciplinaires sur les Peptides (IFRMP) 23, Institut National de la Santé et de la Recherche Médicale (INSERM) U413, Department of Endocrinology, Hospital of Boisguillaume, Centre Hospitalier Universitaire of Rouen, 76031 Rouen Cedex, France. E-mail: herve.lefebvre{at}chu-rouen.fr.
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Abstract |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The presence of ectopic GIP receptors in food-dependent adrenal lesions causing Cushing’s syndrome has been demonstrated by in vitro incubation, in situ hybridization, and RT-PCR studies (7, 8, 9, 10). Similarly, overexpression of the mRNAs encoding the eutopic V1a and 5-HT4 receptors has been described recently in tissue explants removed from AIMAHs responsive in vivo to AVP and cisapride, respectively (11, 12, 13, 14). The normal human adrenal cortex contains substantial amounts of AVP and 5-HT, which can enhance cortisol secretion through paracrine mechanisms (15, 16, 17). Thus, it is conceivable that these factors may stimulate cortisol secretion by AIMAHs via activation of the abnormally expressed V1a and 5-HT4 receptors. However, the occurrence of AVP and 5-HT in hyperplastic adrenal tissues and the sensitivity of AIMAH cells to AVP and 5-HT, in comparison with normal adrenocortical cells, have never been investigated.
The aim of the present study was to evaluate in vivo and in vitro the responsiveness of AIMAH to various bioactive signals to detect the expression of illegitimate receptors by the tissues and, consecutively, determine the contribution of illegitimate receptors to the pathophysiology of the disease. For this purpose, four consecutive AIMAH patients with adrenal Cushing’s syndrome systematically underwent a series of physiological and pharmacological tests before adrenalectomy. After surgery, in vitro studies were conducted to examine the effects of the regulatory factors that were potentially involved in the abnormal in vivo cortisol responses on glucocorticoid production by cultured cells derived from hyperplastic tissues, in comparison with normal adrenocortical cells. In addition, the possible occurrence of 5-HT and AVP within the hyperplastic tissues abnormally sensitive to these regulatory signals was investigated by immunohistochemistry.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The diagnosis of ACTH-independent Cushing’s syndrome was based on the results of hormonal investigations as reported previously (18, 19, 20, 21). Briefly, an increase in 24-h urinary cortisol excretion, alteration of plasma cortisol circadian rhythm, lack of cortisol suppression under a low-dose dexamethasone test (2 mg/d for 2 d), and suppression of basal plasma ACTH levels [<5 pg/ml (1 pmol/liter)] were observed in the four patients.
Patients
Four consecutive patients with AIMAH responsible for Cushing’s syndrome were investigated.
Patient 1, a 34-yr-old woman, was referred for diabetes mellitus, hypokalemia (3.4 mmol/liter), and hypertension (blood pressure, 180/100 mm Hg). She had hypertension during each of three full-term pregnancies, occurring respectively 6, 5, and 1.5 yr earlier. Gestational diabetes mellitus was also detected during the third pregnancy and treated with insulin. Physical examination revealed faciotruncal adiposity. Menstrual cycles were regular. ACTH-independent Cushing’s syndrome was evidenced by hormonal investigations (Table 1
). Abdominal computerized tomography (CT) showed multinodular enlargement of both adrenal glands that reached 4 cm in diameter on the right and 6 cm in diameter on the left.
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). The analysis of circadian cortisol rhythm was highly suggestive of food-dependent Cushing’s syndrome. Abdominal CT scan revealed multinodular enlargement of the two adrenal glands that reached 3.5 cm in diameter on the right and 4 cm in diameter on the left.
Patient 3, a 49-yr-old woman, was referred for a recent disequilibrium of a 10-yr duration diabetes mellitus. She had two normal pregnancies at the ages of 23 and 25 yr. She also had a history of hypertension for 10 yr and heart failure. She presented with proximal myopathy, large ecchymotic lesions, facial erythrosis, hirsutism, faciotruncal adiposity, and spaniomenorrhea. ACTH-independent Cushing’s syndrome was demonstrated by hormonal investigations (Table 1). Abdominal CT scan showed a multinodular enlargement of both adrenal glands that reached 5 cm in diameter on the right and 5.5 cm in diameter on the left.
Patient 4, a 54-yr-old woman, was referred for clinical signs of Cushing’s syndrome including large ecchymotic lesions and central obesity, with a 12-kg weight gain during the last year. She had hypertension, which was treated for 18 months, and a 2.5-yr history of unexplained increase in circulating neutrophils. Physical examination revealed mild facial hirsutism, proximal myopathy, and skin thinning. She had undergone hysterectomy without oophorectomy at the age of 42 because of uterine fibromas. She had two normal pregnancies at the ages of 29 and 31 and an extrauterine pregnancy at the age of 39. ACTH-independent Cushing’s syndrome was evidenced by hormonal investigations (Table 1). The analysis of circadian cortisol rhythm was highly suggestive of food-dependent Cushing’s syndrome. Abdominal CT scan revealed bilateral macronodular adrenal hyperplasia that reached 5 cm in diameter on the right and 5.4 cm in diameter on the left.
All patients underwent bilateral laparoscopic adrenalectomy, and macronodular adrenocortical hyperplasia was confirmed by pathological examination.
Clinical investigation protocol
Potentially illegitimate membrane hormone receptors were systematically searched for, after informed consent of the patients, by using a modified version of the clinical protocol previously published by Lacroix et al. (1). The study was approved by the institutional ethics committee. It was performed on 4 consecutive days after the initial routine hormonal investigations. Briefly, PCL were measured in response to a posture test, a standard mixed meal, administration of 250 µg ACTH1–24 iv used as a reference test, combined iv administration of 100 µg GnRH and 250 µg TRH, 10 mg cisapride (Prépulsid, Janssen-Cilag Laboratories, Boulogne-Billancourt, France) orally or 10 mg metoclopramide (Primpéran, Synthélabo Laboratories, Meudon-la-Forêt, France) iv, 1 mg glucagon im, and 0.5 mg terlipressin (a precursor of lysine vasopressin; Glypressine, Ferring Laboratories, Gentilly, France) iv. In patients 2 and 4, TRH and GnRH tests were repeated separately to investigate the cortisol response to the combined TRH/GnRH stimulation test. Patients 2 and 4 also underwent human chorionic gonadotropin (hCG) (5000 IU im) (Gonadotrophine Chorionique Endo, Organon, Eragny-sur-Epte, France) and recombinant human FSH (150 IU im) (Gonal-f, Serono, Boulogne, France) stimulation tests. The four patients studied were maintained in a recumbent position during all pharmacological tests. Plasma cortisol and ACTH assays were performed as described previously (18, 19, 20, 21). In accordance with previous reports (2, 3, 4), a cortisol response was considered nonsignificant when less than 25%, potentially significant when above 25%, and highly significant when above 50% of basal level.
In vitro studies
Tissue collection. Adrenocortical tissues were obtained at surgery and immediately dissected by the pathologist. Normal adrenal explants (control tissues) were obtained from patients undergoing expanded nephrectomy for kidney cancer. Adrenocortical fragments were either transported to the laboratory in culture medium for primary culture and perifusion experiments or fixed in formalin and embedded in paraffin for immunohistochemical analysis. The protocol of collection of the tissues and the experimental procedures were approved by the regional ethics committees, and written informed consent was obtained from all subjects.
Reagents. Protease, collagenase (type IA), deoxyribonuclease I, insulin, apo-transferrin, L-ascorbic acid, 5-HT, isoproterenol, AVP, angiotensin II (Ang II), and the rabbit 5-HT antiserum were purchased from Sigma (St-Quentin Fallavier, France). The nutrient medium F-12 (Ham’s) and DMEM were obtained from Life Technologies, Inc. (Paisley, Scotland, UK). The antibiotic-antimycotic solution and fetal bovine serum were from Bio-Whittaker (Walkersville, MD). GIP and GLP-1-(7–36) amide were from Bachem (Budendorf, Switzerland). GnRH and ACTH were from Ferring SAS (Gentilly, France). hCG was from Organon, and recombinant human FSH was from Serono.
Cell culture. Hyperplasia and normal adrenal gland fragments were immersed in culture medium (50% DMEM to 50% Ham’s F-12) supplemented with 0.2% antibiotic-antimycotic solution and rapidly transported to the laboratory. The adrenal cortex was dissected from fat and medullary tissues and minced with scissors, and adrenal cells were enzymatically dispersed, as described previously (22). Briefly, tissue samples were stirred for 45 min at 37 C in culture medium containing protease (2 mg/ml), collagenase (2 mg/ml), and desoxyribonuclease I (70 µg/ml) in a 5% CO2-95% air atmosphere. The tissues were disaggregated by gentle aspirations with a sterile 10-ml pipette. Dispersed cells were filtered on a nylon sieve (100-µm mesh opening). The cell suspension was centrifuged (100 x g, 37 C, 20 min). Residual tissue fragments were subjected to a second period of digestion/dispersion procedure as described above. Isolated cells were then transferred into culture medium supplemented with 5 µg/ml insulin, 10 µg/ml apo-transferrin, 20 mg/ml ascorbic acid, and 5% fetal calf serum. Adrenocortical cells were cultured in petri dishes (at a density of 106 cells per dish) and incubated at 37 C in a 5% CO2-95% air atmosphere with 100% relative humidity. The culture medium was changed 24 h after plating. Incubation experiments of cells with 5-HT were conducted after 2 d in culture with fresh DMEM (control) or DMEM containing different concentrations of GIP, GLP-1, hCG, GnRH, AVP, 5-HT, ACTH, Ang II, or isoproterenol. Cells were incubated with each secretagogue for 24 h at 37 C. After the incubation period, aliquots of the culture medium were taken and immediately frozen at –20 C until cortisol RIA. Results are expressed as mean ± SE, and statistical significance was assessed by Bonferroni test after one-way ANOVA.
Perifusion experiments. Hyperplasia tissues obtained at surgery from patients 2 and 4 were studied by use of a perifusion system technique as described previously (16). Briefly, hyperplasia fragments were dipped into 100 ml DMEM and rapidly transported to the laboratory. The adrenocortical explants were diced into small pieces (1–2 mm3), rinsed three times with fresh medium, mixed with biogel P2, and transferred into perifusion chambers. The tissue fragments were perifused with DMEM at constant flow rate (300 µl/min), pH (7.4), and temperature (37 C). The perifusion medium was continuously gassed with a 95% O2-5% CO2 mixture. The tissues were allowed to stabilize for 2 h before any test substance was administered. Test substances were dissolved in gassed DMEM and infused into the perifusion chambers at the same flow rate as DMEM alone, by means of a multichannel peristaltic pump. Fractions of the effluent perifusate were collected every 5 min and immediately frozen until assay. Cortisol levels were determined in all fractions by RIA, as described previously (16).
Immunohistochemistry. Deparaffinized sections from hyperplasia tissues were incubated overnight at 4 C in a humidified atmosphere, with polyclonal rabbit antibodies directed against either 5-HT (1:400) or AVP (1:400) (provided by Dr. A. Burlet, Nancy University, Nancy, France). The sections were then incubated with a streptavidin-biotin-peroxidase complex (Dako Corporation, Carpinteria, CA), and the enzymatic activity was revealed with 3-amino-ethylcarbazole. The specificities of the immunoreactions were controlled by using 5-HT- or AVP-antisera preabsorbed for 2 h at 20 C with 10–6 M 5-HT or 10–6 M AVP, respectively. The tissue sections were counterstained for 1 min with hematoxylin, mounted in Eukitt (Kindler Gmbh & Co., Freiburg, Germany), coverslipped, and examined on an Eclipse E600 microscope (Nikon, Les Ulis, France) equipped with a charge-coupled device DXC-950 camera (Sony, Paris, France).
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Results |
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Effect of food intake on cortisol secretion
In patients 2 and 4, the mixed meals induced a robust increase in plasma cortisol (Table 2 and Fig. 1A). The plasma circadian cortisol rhythm was suggestive of food-dependent Cushing’s syndrome in both cases (data not shown), although in patient 4, fasting PCL remained relatively high (Table 1). In contrast, mixed meals had no influence on plasma cortisol in patients 1 and 3. Incubation of cultured adrenocortical cells obtained from patients 2 and 4 with increasing concentrations of GIP (10–10 to 10–6 M) provoked a dose-dependent stimulation of cortisol secretion, whereas administration of GLP-1 (10–10 to 10–6 M) had no effect (Fig. 1B). GIP was equally potent to stimulate steroidogenesis from patient 2 hyperplasia (H2) and patient 4 hyperplasia (H4) cells (pEC50 were 9.6 ± 0.3 and 10.2 ± 0.2, respectively; mean ± SE, n = 4), but was more efficient to stimulate cortisol production from H2 cells (Emax = +320 ± 31%; n = 4) than H4 cells (Emax = +211 ± 8%; n = 4; P < 0.01). The effect of GIP (10–7 M) on cortisol secretion was also evaluated in adrenocortical cell cultures derived from patients 1 and 3 who did not respond to food intake. Surprisingly, GIP induced a +106% increase in cortisol secretion from H1 cells, whereas it had no affect on H3 cells. Administration of GIP (10–7 M; 20 min) to perifused H2 explants induced a massive but transient increase in cortisol release that reached a maximum of +854 ± 189% (n = 3; Fig. 1C).
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A significant increase in PCL was observed in patients 2 and 4 (+463 and +194%, respectively) after combined iv administration of TRH (250 µg) and GnRH (100 µg), whereas no cortisol response was observed in patients 1 and 3 (Table 2). Additional investigations performed in patients 2 and 4 revealed that iv injection of GnRH alone (100 µg) induced a significant increase in PCL reaching +99 and +254%, respectively (Fig. 2A). In patient 2, plasma LH concentration increased from less than 0.1 units/liter to 31 units/liter during the combined TRH/GnRH test, whereas it rose from 1.4 to 18 units/liter after GnRH injection. In the two patients, hCG (5000 IU im) also stimulated cortisol secretion (+342% in patient 2, and +101% in patient 4; Fig. 2A), whereas neither recombinant FSH (150 IU sc) nor TRH (250 µg iv) had any influence on PCL (data not shown). Incubation of cultured adrenocortical H2 and H4 cells with hCG (10–10 to 10–6 M) provoked a dose-dependent stimulation of corticosteroidogenesis (Fig. 2B). The pEC50 and efficacy (Emax) of hCG to stimulate cortisol production were 9.3 ± 0.4 and +243 ± 41%, respectively, in H2 cells and 9.3 ± 0.1 and +40 ± 2% in H4 cells (n = 4). In case 2, the maximum effect of hCG was obtained at a concentration of 10–8 M, and attenuation of the response was observed at higher concentrations of hCG (10–7 and 10–6 M). For concentrations up to 10–6 M, hCG had no effect on cortisol secretion from normal adrenocortical cells (Fig. 2B). Similarly, GnRH did not affect the secretory activity of H2 and H4 cells (Fig. 2B). The kinetics of the responses of perifused H2 and H4 explants to a pulse of hCG (10–6 M; 20 min) is shown in Fig. 2C. In both cases, hCG induced a rapid increase in cortisol secretion (+1300% for H2 cells and +507% for H4 cells), followed by a plateau phase with spontaneous oscillations of the secretion rate.
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In patient 1, administration of cisapride (10 mg, orally) induced a significant increase in PCL (Table 2). A similar cortisol response was observed after a metoclopramide stimulation test (10 mg iv) performed during the follow-up of the patient (Fig. 3A). No stimulation of cortisol secretion was detected in patients 2 and 4 after administration of 5-HT4 receptor agonists (Table 2). Incubation of H1 cells with 5-HT (10–9 to 10–5 M) induced a dose-dependent stimulation of cortisol production (Fig. 3B). The potencies of 5-HT to stimulate corticosteroidogenesis in H1 cells and normal adrenocortical cells were similar (7.0 ± 0.1 and 7.3 ± 0.4, respectively; n = 4), but the efficacy of 5-HT to stimulate cortisol production was higher in H1 cells (+173 ± 6%) than in normal adrenocortical cells (+93 ± 12%; P < 0.0001).
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A significant increase in PCL was observed during upright stimulation test in patients 1, 2, and 3 (Table 2 and Fig. 4A). Administration of the vasopressin analog terlipressin (0.5 mg iv) also stimulated cortisol secretion in patient 1 but not in patients 2 and 4 (Table 2 and Fig. 4A, inset). Incubation of Ang II (10–7 M) with H1, H2, H4, and normal adrenocortical cells induced a significant increase in cortisol secretion (Fig. 4B). The amplitude of the stimulation was significantly higher in cells derived from the hyperplastic tissues (H1 cells, +150 ± 41%; H2 cells, +202 ± 33%; H4 cells, +116 ± 7%) than in normal adrenocortical cells (+59 ± 9%). The ß-adrenoreceptor isoproterenol (10–5 M) did not significantly modify cortisol secretion either from cultured H1, H2, and H3 cells or from normal adrenocortical cells (Fig. 4C). In contrast, incubation of H4 cells with isoproterenol provoked a +72 ± 3% increase in cortisol production. AVP (10–8 M) induced a significant increase in cortisol secretion from all types of cells (Fig. 4D). The amplitudes of the responses of H2 cells (+166 ± 28%), H3 cells (+75 ± 13%), and H4 cells (+51 ± 5%) were in the same range as those of normal cells (+87 ± 35%), whereas the amplitude of the response to AVP was significantly higher in H1 cells (+380 ± 85%). AVP was equally potent to stimulate cortisol production in H1 cells (pEC50 = 10.1 ± 0.1) and in normal cells (pEC50 = 9.7 ± 0.2) (Fig. 4D, inset).
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Incubation of H1 tissue slices with 5-HT antibodies produced intense labeling of the subcapsular region of the cortex (Fig. 5A). Clusters of immunoreactive cells were also observed in the central zones of hyperplastic nodules (Fig. 5B). 5-HT-positive cells had the morphological characteristics of spongiocytic cells, i.e. cells with abundant cytoplasm and numerous lipid droplets. Preincubation of the 5-HT antiserum with 5-HT (10–6 M) totally abolished immunostaining (Fig. 5C). Similarly, labeling of H1 tissue slices with AVP antibodies revealed the presence of immunoreactive material in the spongiocytic cells of the subcapsular region of the cortex (Fig. 5D). Small groups of immunoreactive cells were also detected in the hyperplastic nodules (Fig. 5E). In contrast, no labeling was observed when the AVP antibodies were preincubated with AVP (Fig. 5F).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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20 pmol/liter; Ref.24). Finally, the relatively high fasting PCL observed in patient 4 suggests that other illegitimate receptors than the GIP receptor may play a significant role in the maintenance of cortisol secretion during interprandial periods. The two hyperplasias sensitive to GIP also responded in vivo to combined administration of TRH and GnRH, GnRH alone, and hCG, but not to recombinant human FSH. The higher relative increase in PCL observed after concomitant injection of TRH and GnRH in patient 2, in comparison with the cortisol response induced by GnRH alone, likely resulted from the more pronounced GnRH-induced LH increase. Incubation of adrenocortical cells with hCG induced a dose-dependent increase in cortisol production, whereas GnRH had no effect on steroidogenesis in both cases. Thus, this study provides the first observation of AIMAHs responding to both LH/hCG and GIP. Conversely, the two tissues were unresponsive to 5-HT4 receptor agonists, showing that an abnormal cortisol response to LH/hCG is not always associated with an increased sensitivity of AIMAH to serotonergic drugs, as suggested in a recent report (25). Consistent with a previous study showing that, in the human adrenal cortex, the LH/hCG receptor is almost exclusively expressed by zona reticularis (26), we did not observe any effect of hCG on cortisol production by normal adrenocortical cells. In one hyperplasia, the attenuation of the cortisol response observed with high concentrations of hCG suggests the occurrence of a desensitization phenomenon of the receptor, as previously shown in Leydig cells (27, 28). The molecular mechanisms involved in the prolonged stimulation of cortisol production evoked by a single pulse of hCG in perifused hyperplasia fragments remain unclear. In addition to its direct effect on steroidogenic cells, hCG may activate intraadrenal regulatory systems, which may in turn stimulate cortisol secretion.
It has previously been shown that the 5-HT4 receptor, which is physiologically expressed in the human adrenal cortex, mediates the stimulatory effects of 5-HT on aldosterone and cortisol production (16, 29, 30, 31). It has also been observed that 5-HT4 receptor mRNAs are overexpressed in most cisapride/metoclopramide-responsive AIMAHs causing subclinical and/or overt Cushing’s syndrome (12). In agreement with these data, we found that the abnormal cortisol response to cisapride observed in one patient was associated with an increased efficacy of 5-HT to stimulate cortisol production in vitro from hyperplasia cells, in comparison with normal adrenocortical cells. The presence of 5-HT-like immunoreactivity, in a subpopulation of spongiocytic cells within the hyperplastic cortex, suggests that cortisol production may be under the control of an intraadrenal serotonergic tone involving an autocrine/paracrine mode of regulation. The occurrence of 5-HT in corticosteroidogenic cells has been previously reported in the rat adrenal gland, where glomerulosa cells are able to synthesize significant amounts of 5-HT through uptake and decarboxylation of circulating 5-hydroxytryptophan (32). However, in the normal human adrenal gland, 5-HT is exclusively stored in perivascular mast cells (16). Therefore, the present study provides the first demonstration of the ectopic localization of a paracrine regulatory factor, i.e. 5-HT, in a pathological adrenal tissue.
Three AIMAHs were shown to respond in vivo to the posture test. The increase in cortisol levels induced by a change from supine to upright posture may involve several hormones, including Ang II, catecholamines, and AVP (1). Thus, we have investigated in vitro the effect of these signals on cortisol production in cultured cells derived from the four tissues and from normal adrenocortical cells. As previously shown (17, 33, 34), the secretory activity of normal cells was stimulated by Ang II and AVP but was not influenced by the ß-adrenoreceptor agonist isoproterenol. The data obtained in vitro from hyperplasia tissues were not strictly correlated with those of clinical testing. For instance, the in vitro sensitivities of the posture-responsive hyperplasia H3 to Ang II, isoproterenol, and AVP were similar to those of the normal adrenocortical tissue, whereas hyperplasia H4, which did not respond in vivo to the posture test, exhibited exaggerated in vitro responses to Ang II and isoproterenol. However, in hyperplasias 1 and 2, the results of cell incubation experiments clearly showed that the in vivo cortisol response observed during the upright stimulation test was mediated by Ang II, as previously suggested by Nakamura et al. (35), after in vivo pharmacological studies in a patient with posture-sensitive AIMAH. Similarly, the increased efficacy of AVP to stimulate cortisol production in H1 cells, in comparison with normal cells, which likely reflects an overexpression of vasopressin V1a receptor mRNAs by the hyperplastic tissue (11), explains the abnormal in vivo cortisol response of patient 1 to terlipressin. Considering the low physiological plasma concentrations of AVP (10–12 M; Ref.36), it is unlikely that circulating AVP may influence cortisol production from hyperplasia cells. Therefore, if AVP were to play a significant role in the pathophysiology of cortisol hypersecretion in AIMAH, the nonapeptide would have to be produced locally. The immunohistochemical results obtained from hyperplasia 1 confirmed the presence of AVP-like immunoreactivity in the tissue, consistent with a previous study showing the occurrence of proAVP mRNA in AIMAHs (11). Contrary to the normal adrenal gland, where AVP is stored in chromaffin cells (17), the hyperplastic cortex exhibited AVP-like immunoreactivity in a subpopulation of steroidogenic cells having the same tissular distribution as that of serotonergic cells. The detection of 5-HT and AVP in AIMAH spongiocytic cells provides additional evidence for the presence of hybrid cells, i.e. exhibiting both steroidogenic and neuroendocrine cell characteristics, in adrenocortical cortisol-producing lesions (23). It also indicates that AVP, like 5-HT, can act as an ectopic autocrine/paracrine regulatory factor in some AIMAH tissues. The role of this paracrine mode of communication in the maintenance of active corticosteroidogenesis will be explored in vitro and in vivo, by means of perifusion and clinical studies with specific vasopressinergic and/or serotonergic antagonists.
In conclusion, the present study additionally demonstrates that hyperplastic ACTH-independent adrenocortical tissues may simultaneously express multiple illegitimate membrane receptors for both circulating and intraadrenal paracrine/autocrine factors. In particular, we describe for the first time adrenocortical hyperplasias responding to both gonadotropin and GIP. Our data also demonstrate a hyperresponsiveness of AIMAH cells to 5-HT, Ang II, and AVP, in comparison with normal adrenocortical cells. Finally, they show for the first time that adrenal hyperplasias produce corticotropic substances on their own, providing evidence for autocrine/paracrine regulations of cortisol secretion within the tissues.
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Acknowledgments |
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Footnotes |
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First Published Online December 7, 2004
Abbreviations: AIMAH, ACTH-independent macronodular adrenal hyperplasia; Ang II, angiotensin II; AVP, arginine vasopressin; CT, computerized tomography; GIP, gastric inhibitory polypeptide; GLP-1, glucagon-like peptide-1; hCG, human chorionic gonadotropin; 5-HT, serotonin; PCL, plasma cortisol level(s).
Received June 30, 2004.
Accepted November 24, 2004.
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References |
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-hydroxylase, and 3 ß-hydroxysteroid-dehydrogenase messenger ribonucleic acid and proteins and on steroidogenic responsiveness to corticotropin and angiotensin-II. J Clin Endocrinol Metab 78:1212–1219[Abstract]
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A. Lampron, I. Bourdeau, P. Hamet, J. Tremblay, and A. Lacroix Whole Genome Expression Profiling of Glucose-Dependent Insulinotropic Peptide (GIP)- and Adrenocorticotropin-Dependent Adrenal Hyperplasias Reveals Novel Targets for the Study of GIP-Dependent Cushing's Syndrome J. Clin. Endocrinol. Metab., September 1, 2006; 91(9): 3611 - 3618. [Abstract] [Full Text] [PDF]
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G. Abiven, J. Coste, L. Groussin, P. Anract, F. Tissier, P. Legmann, B. Dousset, X. Bertagna, and J. Bertherat Clinical and Biological Features in the Prognosis of Adrenocortical Cancer: Poor Outcome of Cortisol-Secreting Tumors in a Series of 202 Consecutive Patients J. Clin. Endocrinol. Metab., July 1, 2006; 91(7): 2650 - 2655. [Abstract] [Full Text] [PDF]
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L. Groussin, A. Horvath, E. Jullian, S. Boikos, F. Rene-Corail, H. Lefebvre, F.-L. Cephise-Velayoudom, M.-C. Vantyghem, P. Chanson, B. Conte-Devolx, et al. A PRKAR1A Mutation Associated with Primary Pigmented Nodular Adrenocortical Disease in 12 Kindreds J. Clin. Endocrinol. Metab., May 1, 2006; 91(5): 1943 - 1949. [Abstract] [Full Text] [PDF]
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 P. A. Nepomnaschy, K. B. Welch, D. S. McConnell, B. S. Low, B. I. Strassmann, and B. G. England Cortisol levels and very early pregnancy loss in humans PNAS, March 7, 2006; 103(10): 3938 - 3942. [Abstract] [Full Text] [PDF]
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T. L. Mazzuco, O. Chabre, J.-J. Feige, and M. Thomas Aberrant Expression of Human Luteinizing Hormone Receptor by Adrenocortical Cells Is Sufficient to Provoke Both Hyperplasia and Cushing's Syndrome Features J. Clin. Endocrinol. Metab., January 1, 2006; 91(1): 196 - 203. [Abstract] [Full Text] [PDF]
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D. Cartier, S. Jegou, F. Parmentier, I. Lihrmann, E. Louiset, J.-M. Kuhn, C. Bastard, P.-F. Plouin, M. Godin, H. Vaudry, et al. Expression profile of serotonin4 (5-HT4) receptors in adrenocortical aldosterone-producing adenomas Eur. J. Endocrinol., December 1, 2005; 153(6): 939 - 947. [Abstract] [Full Text] [PDF]
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R. Libe and J. Bertherat Molecular genetics of adrenocortical tumours, from familial to sporadic diseases Eur. J. Endocrinol., October 1, 2005; 153(4): 477 - 487. [Abstract] [Full Text] [PDF]
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). B, Effect of graded concentrations of GIP and GLP-1 (from 10–10 to 10–6 M) on cortisol production from cultured cells derived from hyperplasias H2 (•, GIP; and 
, GLP-1) and H4 (
, GLP-1). Inset, Effect of GIP (10–7 M) on cortisol production from cultured cells derived from hyperplasias H1 and H3. C, Effect of a single pulse of GIP (10–7 M, 20 min) on cortisol production from perifused hyperplasia H2 explants. The spontaneous level of cortisol release (100% basal level) was calculated as the mean of the eight consecutive fractions preceding the pulse of GIP.
, hCG). C, Effect of a single pulse of hCG (10–6 M, 20 min) on cortisol production from perifused adrenocortical explants removed from hyperplasias H2 (•) and H4 (
) and normal adrenocortical cells (
, P < 0.05; 