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Cushing’s Syndrome in a Patient with Bilateral Macronodular Adrenal Hyperplasia Responding to Cisapride: An in Vivo and in Vitro Study

Endocrine Unit, Department of Clinical Physiopathology, University of Florence, 50139 Florence, Italy

Address all correspondence and requests for reprints to: Massimo Mannelli, M.D., Endocrine Unit, Department of Clinical Physiopathology, University of Florence, Viale Pieraccini, 6, 50139 Florence, Italy. E-mail: m.mannelli{at}dfc.unifi.it.

	Abstract

Top Abstract Introduction Patient and Methods Results Discussion References

 
Cortisol secretion in adrenal Cushing’s syndrome can be regulated by aberrant hormone receptors, such as gastric inhibitory polypeptide, V1 vasopressin, catecholamines, LH/human chorionic gonadotropin, and serotonin receptors. We report the case of a patient with Cushing’s syndrome due to bilateral adrenal macronodular hyperplasia. Extensive in vivo testing for the presence of aberrant receptors revealed a 5-fold increase of plasma cortisol after the administration of cisapride, an agonist of the serotonin 4 (5-HT₄) receptor. Primary cell cultures were established from adrenocortical specimens obtained at surgery, and in vitro studies also showed that cisapride determined an increase [133.7 ± 5.5% (mean ± SE) of baseline, considered 100%) of cortisol secretion from cultured cells. The presence of 5-HT₄ receptor transcript, and in particular of isoforms c, g, and n, was confirmed by RT-PCR, and the determination of the mRNA levels by real-time RT-PCR revealed a higher expression than in normal adrenal glands. To our knowledge, this is one of the first reports of Cushing’s syndrome in which cortisol secretion is regulated mainly by the 5-HT₄ receptor, among known aberrant receptors. In addition, it is noteworthy that hypocortisolism ensued after the removal of the most enlarged adrenal gland, but the in vivo response to cisapride persisted.

	Introduction

Top Abstract Introduction Patient and Methods Results Discussion References

 
ENDOGENOUS CUSHING’S SYNDROME is attributable to a primary adrenal pathology in about 20% of cases (1, 2). Among these, bilateral adrenal hyperplasia is responsible for approximately 10% of the syndrome. Mutations of the gene encoding the protein kinase A type I- regulatory subunit have been demonstrated recently in patients with bilateral pigmented micronodular adrenal hyperplasia (3). Although the pathophysiology of endogenous hypercortisolism, especially in cases of ACTH-independent unilateral adrenal adenoma or bilateral macronodular adrenal hyperplasia (AIMAH), has been only partially unraveled until recently (1, 2), it is undoubtedly becoming better understood. AIMAH is very often characterized by the presence of illicit hormone receptors on the adrenocortical cells, as suggested by the abnormal in vivo cortisol secretion evoked by substances that do not cause any plasma cortisol increase in normal subjects (4). Several types of adrenal illicit receptors have been described in primary adrenal Cushing’s syndrome; among these are receptors for gastric inhibitory polypeptide, V1-vasopressin receptors, ß-adrenergic receptors, LH/human chorionic gonadotropin (hCG) receptors, and serotonin 4 (5-HT₄) receptors (4). Adrenal illicit 5-HT₄ receptors have been found, by means of cortisol response to cisapride or metoclopramide in patients with AIMAH and subclinical or overt Cushing’s syndrome, in association with other aberrant receptors (i.e. LH/hCG, V1-vasopressin, ß-adrenergic receptors) (5, 6, 7, 8). In only one patient has the exclusive presence of the 5-HT₄ receptor, among known illicit adrenal receptors, been described so far (7). The demonstration that the presence and activation of adrenal illicit receptors may be responsible for chronic hypercortisolism has been provided in some cases in which remission of the disease was observed under specific receptor blockade (5, 9). With regard to the 5-HT₄ receptor, it is worth mentioning that nine different isoforms have been described so far (10, 11, 12, 13), and in a recent study isoform characterization and quantitative determination of 5-HT₄ receptor mRNA in AIMAH have been performed (7). Interestingly, 5-HT₄ receptor overexpression was found in four of six cases. On the other hand, in the two remaining cases, the expression level was similar to that observed in the normal adrenal cortex, and isoforms present in normal glands were also detected. Furthermore, no gain-of-function mutations of the 5-HT₄ receptors were found in these two cases in which the enhanced sensitivity to 5-HT₄ receptor agonists remains unclear (7). In this paper, we report the case of a patient affected by Cushing’s syndrome due to AIMAH who showed a clear increase in plasma cortisol levels only after cisapride administration during the screening for illicit receptors, suggesting at first sight the involvement of 5-HT₄ in the pathogenesis of her disease.

	Patient and Methods

Top Abstract Introduction Patient and Methods Results Discussion References

 
Patient

A 58-yr-old woman was referred to our unit for a clinical syndrome characterized by weight gain, fatigue, muscle weakness, and hypertension. An abdominal ultrasound scan, performed after the finding of an altered value of -glutamyl transferase, had revealed the presence of bilateral adrenal enlargement.

The clinical history of the patient was characterized by a hysterectomy performed after her third pregnancy, when she was 36 yr old, and by frequent episodes of bronchial asthma and an external quadrantectomy of her right mammary gland for a differentiated ductal carcinoma (G1 Bloom-Richardson), performed 4 months before her admission. For this disease, the patient was receiving local radiotherapy and medical treatment with tamoxifen when evaluated.

Physical examination revealed central obesity with cervical buffalo hump, a rounded face, thin skin with bruises, a marked dorsal scoliosis, and marked muscle hypotrophy of the limbs. Body weight was 63 kg, height was 158 cm, blood pressure was 170/100 mm Hg, and heart rate was 100 beats per minute.

Initial investigation showed an increase in urinary free cortisol, with 444 and 373 nmol/24 h on 2 different days (normal values, <275 nmol/24 h). Circadian rhythm of cortisol was absent, as shown by the following values: 671 nmol/liter at 0800 h, 668 at 1200 h, 721 at 1600 h, 774 at 2000 h, and 656 at 2400 h. Basal plasma ACTH was 10.5 ng/liter (normal range, 9–52 ng/liter). Plasma concentration of dehydroepiandrosterone sulfate was below 0.4 µmol/liter (normal range, 0.4–5.4 µmol/liter); testosterone was 1.7 nmol/liter (normal range, 0.5–3.0 nmol/liter); -4-androstenedione, 1.4 nmol/liter (normal range, 1.4–9.4 nmol/liter); and 17-hydroxy-progesterone, 0.52 nmol/liter (normal range, 1.2–8.0 nmol/liter). Blood electrolytes and fasting and postprandial glucose were normal, triglycerides were elevated (350 mg/dl; normal range, 50–170 mg/dl). Plasma ACTH and cortisol were not suppressed after an overnight administration of either 2 mg (10.6 ng/liter and 501 nmol/liter, respectively) or 8 mg (10.4 ng/liter and 875 nmol/liter, respectively) dexamethasone.

An abdominal computed tomography (CT) scan confirmed the bilateral adrenal macronodular hyperplasia with a predominant 7.4 x 3.9-cm solid, hypodense nodule in the left gland and a similar 4.0 x 2.1-cm nodule in the right gland. Hounsfield units were 18 on unenhanced CT scan and 32 on enhanced CT scan.

An ¹³¹I-cholesterol scan showed an intense uptake of the tracer after 24 h in the left adrenal and a less intense uptake after 48 h in the contralateral gland.

After 1 month of therapy with ketoconazole (200 mg twice a day), the patient had her left adrenal surgically removed by laparoscopy.

One week, 4 months, and 16 months after surgery the patient was again admitted to our unit for further evaluation.

Investigation protocol

The patient was studied according to a protocol that had been approved by the local ethical committee and by the patient, with a written informed consent. Studies were performed after an overnight fast, with the patient in the supine position for at least 60 min before testing. The protocol to screen for potential illicit adrenal receptors included serial measurement of cortisol and other hormones at different intervals during various tests, which were performed sequentially over the course of several days and according to the protocol suggested by Lacroix et al. (4). A change in plasma cortisol less than 25% from baseline (100%) was considered as no response, according to Mircescu et al. (6), a 25–49% change was considered a partial response, and a change of 50% or greater was considered a positive response. Tests included the administration of 100 µg GnRH iv (Relefact, Hoechst-Roussel, Montreal, Canada); 200 µg TRH iv (Relefact-TRH, Hoechst-Roussel); 2.5 µg desmopressin sc (Minirin, Ferring Pharmaceuticals Ltd., North York, Ontario, Canada); 10 IU arginin-vasopressin (AVP) im (Pitressin, Goldshield Pharmaceuticals, Croydon, UK); 1 mg glucagon iv (Eli Lilly, Indianapolis, IN); 10 mg cisapride orally (Prepulsid, Janssen Pharmaceuticals, Titusville, NJ); and 250 µg ACTH-(1–24) iv (Cortrosyn, Organon Inc., West Orange, NJ). Other tests included a standard mixed meal (517 kcal, 48% carbohydrates, 38% lipids, 14% proteins) and a posture test performed in a 2-h supine position followed by a 2-h ambulation period. A stimulation test using cisapride was repeated twice before and three times after surgery. ACTH stimulation test was performed before and after surgery.

Assays

Cortisol (plasma, urine, culture medium) was measured by an electrochemiluminescent assay (Roche Diagnostics, Monza, Italy). ACTH was measured by a chemiluminescent assay (Nichols Institute Diagnostics, San Clemente, CA).

Cell cultures

Adrenocortical fragments obtained at surgery were processed for cell preparation or frozen in liquid nitrogen and stored at -80 C. A large fragment was sent to the Pathology Department for histopathology that assessed the presence of a pattern of macronodular adrenal hyperplasia. Primary cell cultures were established within 1 h of surgery, as described by Munari-Silem et al. (14), with some modifications. Briefly, the tissue was freed of fat, minced, and incubated for 20 min at 37 C in PBS (Sigma Chemical Co., St Louis, MO) containing 2 mg/ml collagenase (Sigma Chemical Co.). To facilitate dispersion, tissue was minced with a Pasteur pipette with a fine heat-polished tip; the cell suspension was then filtered through a cell strainer (80-µm mesh, Sigma Chemical Co.) and centrifuged 10 min at 1400 rpm. The pellet was resuspended in a culture medium consisting of a 1:1 (vol/vol) mixture DMEM/F-12 with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, and enriched with a mixture of insulin/transferrin/selenium (Sigma Chemical Co.). Isolated cells were plated onto 35-mm diameter culture dishes at a density of 50–70 x 10³ cells per dish and cultured at 37 C in 95% air/5% CO₂ in a fully humidified environment. Primary cultures were used within 3–4 d. The precise number of cells used in each experimental protocol was determined at the end of the experiments using a hemocytometer.

In vitro cortisol secretion studies

Cells were seeded in six-well plates. After 3–4 d of culture, the growth medium was removed, and the cells were accurately washed in PBS and incubated in phenol red- and serum-free medium containing 0.1% BSA. After 24 h, ACTH-(1–24) (1 nM), cisapride (1 µM), or a combination of ACTH-(1–24) (1 nM) and cisapride (1 µM) was added to the cultures. After 24 h, the medium was harvested and kept frozen at -20 C in aliquots (300–400 µl) until steroid hormone determination. Cells in phenol red- and serum-free medium containing 0.1% BSA were used as basal controls. After the 24-h harvesting, cells were trypsinized and counted using a hemocytometer. In the same experiment, each point was repeated in duplicate. The experiments were repeated twice. Cortisol secretion (picomoles per 10⁶ cells) was expressed as percentage (mean ± SE) of the cortisol secretion in unstimulated cells (control).

RT-PCR for 5-HT₄, ß1-adrenergic, and angiotensin II type 1 receptors

RT-PCR was performed on total RNA (1 µg for each reaction) extracted from a frozen tumoral fragment, according to the method described by Chomczynski and Sacchi (15). In addition, RNA extracted from normal adrenal glands, obtained during nephrectomy for renal carcinomas, normal human kidney, and myometrium, was used in RT-PCR experiments. A commercially available kit (SuperScript One Step RT-PCR System, Stratagene, La Jolla, CA) was used to prepare the mixture for RT-PCR. For the detection of 5-HT₄ receptor, mRNA primers and experimental conditions were as described previously by Lefebvre et al. (16). For the detection of angiotensin II type 1 (AT-1) receptors, specific primers were designed using the proprietary software Primer Express (Applied Biosystems Inc., Foster City, CA). The expression of ß1-adrenergic receptors was investigated using specific primers, as described previously (17). The PCR products were subjected to agarose gel electrophoresis, blotted on a nylon membrane (in the case of 5-HT₄ receptor), and hybridized to a digoxigenin-labeled internal oligonucleotide (16). The hybridized cDNAs were detected by using an immunochemiluminescent method (Roche Diagnostics), as described previously (18). The primers and the probe were synthesized by MWG Biotech AG (Ebersberg, Germany). Additional RT-PCR was performed using primers specific for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, as described previously (19), to verify that the RNAs were not degraded.

Quantitative determination of 5-HT₄ receptor mRNA

Real-time RT-PCR based on TaqMan technologies for the determination of the levels of 5-HT₄ receptor mRNA was performed using the same primers and probe (labeled with 6-carboxy-fluorescein), as described by Cartier et al. (7), that recognize all 5-HT₄ receptor splice variants. Then, 400 ng total RNA was reverse transcribed in 80 µl of final volume using the TaqMan Universal Master Mix (Applied Biosystems), following the manufacturer’s instructions. The profile of the one-step RT reaction was 10 min at 25 C, 30 min at 48 C, and 5 min at 95 C. The cDNAs were then subjected to PCR, using the following conditions: 2 min at 50 C, 10 min at 95 C, followed by 40 cycles at 95 C for 15 sec and 60 C for 1 min in the ABI Prism 7700 Sequence Detector (Applied Biosystems). A calibration curve was generated using a single-stranded sense oligodeoxynucleotide spanning the sequence included between the primers, as described by Bustin (20). Serial 1:10 dilutions from 1 fg to 100 pg of this oligodeoxynucleotide were subjected to real-time RT-PCR. All the reactions were run in triplicates in the presence of no template controls. Because normalization to GAPDH as well as to other housekeeping genes has been shown not to be very accurate (20), the results were expressed as femtograms of 5-HT₄ receptor mRNA per microgram of total RNA (mean value of three determinations).

Characterization of 5-HT₄ receptor isoforms by RT-PCR

Characterization of 5-HT₄ receptor isoforms was performed as described previously, using primers specific for the different splice variants (7). The PCR products were subjected to agarose gel electrophoresis, blotted on nylon membranes, and hybridized to a digoxigenin-labeled internal oligonucleotide, which served as the probe. The sequence of the oligonucleotide was 5'-CCCTGGGCAGGTGTGGACTGC-3' (Ref.7). The hybridized cDNAs were detected by using an immunochemiluminescent method (Roche Diagnostics), as described previously (18). The primers and the probe were synthesized by MWG Biotech AG (Ebersberg, Germany).

	Results

Top Abstract Introduction Patient and Methods Results Discussion References

 
In vivo evaluation for the presence of illicit adrenal hormone receptors

There was no increase in plasma cortisol after GnRH (LH increased from 4.5 to 11.3 U/liter, and FSH increased from 13.3 to 19.2 U/liter), TRH [TSH increased from 1.07 (normal range, 0.35–3.5) to 8.58 mU/liter; and prolactin increased from 157.2 (normal range, 72–504) to 1085.8 mU/liter], desmopressin, AVP, glucagon, and meal test, whereas a partial response followed the upright posture (Table 1). Conversely, a marked increase, greater than the response observed after ACTH infusion (from 587 to 1843 nmol/liter, 314% of baseline value, considered as 100%) used as a reference test, was observed after cisapride administration (Table 1). Cisapride was administered twice before surgery, and the rise in plasma cortisol was almost superimposable (from 523 to 2658 and from 517 to 2866 nmol/liter, respectively) (Table 1). Baseline urinary concentration of 5-hydroxy indole acetic acid was within the normal range (4.73 mg/24 h; normal range, 2.0–6.0). One week after surgery, the patient was admitted to our unit for further evaluation. At that time she suffered from profound asthenia. Plasma ACTH was 10.5 ng/liter, and plasma cortisol was 94 nmol/liter at 0800 h and 92 nmol/liter at 2400 h. Urinary free cortisol was less than 25 nmol/24 h. Cisapride still caused an evident rise in plasma cortisol levels (from 107 to 664 nmol/liter, 620% of baseline value), and the increase was again greater than the response obtained after ACTH infusion (from 120 to 521 nmol/liter, 434% of baseline value). Cortisone acetate treatment was started (25 mg/d).

View larger version (13K): [in this window] [in a new window]   FIG. 1. Cortisol response to cisapride administration (10 mg orally) before surgery (filled circles, first testing; open circles, second testing) and after surgery (filled squares, 1 wk after surgery; open squares, 4 months after surgery; filled triangles, 16 months after surgery).

Primary cell cultures were established within 1 h of surgery, as described in Patient and Methods. Treatment with ACTH-(1–24) (1 nM), cisapride (1 µM), or a combination of ACTH-(1–24) (1 nM) and cisapride (1 µM) was performed, and after 24 h the medium was harvested for subsequent cortisol determination. Cortisol secretion (picomoles per 10⁶ cells) in the medium was expressed as percentage (mean ± SE) of the cortisol secretion in unstimulated cells (control, 100%). The results, shown in Fig. 2, indicate that ACTH-(1–24) and cisapride, similar to in vivo observations, significantly increased cortisol secretion (171.5 ± 10.3% and 133.7 ± 5.5%, respectively), compared with unstimulated cells. The simultaneous treatment with ACTH-(1–24) and cisapride determined a dramatic increase of cortisol secretion (479.6 ± 68.4%) that was significantly greater than the increase observed with ACTH-(1–24) or cisapride alone. The number of cells (x10⁴) per well did not significantly differ in the treated groups compared with the control group (26.15 ± 3.06, control; 25.68 ± 3.80, ACTH; 24.25 ± 0.75, cisapride; 21.37 ± 1.87, ACTH + cisapride; mean ± SE).

View larger version (17K): [in this window] [in a new window]   FIG. 2. In vitro response to ACTH (1 nM) and/or cisapride (Cis 1 µM), in terms of cortisol secretion.

The presence of the transcript corresponding to 5-HT₄ receptor was investigated by RT-PCR, using specific primers (see Patient and Methods). Total RNA extracted from the excised adrenal gland of the patient was used. In addition, RNA extracted from a normal adrenal gland, obtained during surgery for a renal carcinoma, was subjected to RT-PCR. RNA contamination was excluded by adding a "no RNA" control reaction to the experiment. The expected 346-bp signal was readily detectable after agarose gel electrophoresis from both the tumoral and the normal adrenal gland (Fig. 3A). The identity of 5-HT₄ receptor was confirmed by hybridization with a specific probe, which is derived from an entirely different region of the 5-HT₄ receptor cDNA from the primers (16) (Fig. 3B). In Fig. 3C, the signals corresponding to GAPDH transcript are shown. Because a partial response, in terms of plasma cortisol increase (143% of baseline value, considered as 100%; see Table 1), followed the upright posture, RT-PCR for the detection of AT-1 and ß1-adrenergic receptors was also performed. Specific signals for both AT-1 (251 bp) and ß1-adrenergic (227 bp) receptors were detected from the tumoral as well as from the normal adrenal gland RNA. RNA extracted from a normal kidney served as the positive control for AT-1 receptor, whereas RNA from human myometrium was the positive control for ß1-adrenergic receptor (Fig. 4, A and B).

View larger version (59K): [in this window] [in a new window]   FIG. 3. A, Ethidium bromide-stained gel, showing 5-HT4 receptor-specific RT-PCR products: MW, molecular weight marker VI (Roche Diagnostics); 1, tumoral adrenal sample; 2, normal adrenal gland; 3, no RNA control reaction. B, Hybridization pattern of RT-PCR products from the same experiment as A. C, Ethidium bromide-stained gel, showing GAPDH-specific RT-PCR products.

View larger version (33K): [in this window] [in a new window]   FIG. 4. Ethidium bromide-stained gels, showing AT-1 (A) and ß-1 adrenergic (B) receptor RT-PCR products: MW, molecular weight marker VI (Roche Diagnostics); 1, tumoral adrenal sample; 2, normal adrenal gland; 3, human kidney (A) and human myometrium (B); and 4, no RNA control reaction.

The quantitative expression of 5-HT₄ receptor mRNA was determined from total RNA extracted from the excised adrenal gland of the patient by real-time RT-PCR based on TaqMan technologies (see Patient and Methods) and compared with the expression levels of five normal adrenal glands, obtained during nephrectomy for renal carcinomas. The results are shown in Table 2 and indicate that the highest level of 5-HT₄ receptor mRNA (1.55 fg/µg total RNA) was found in the tumoral gland (no. 1). The mean value of the normal adrenal glands was 0.68 fg/µg total RNA, and in only one case (no. 6) was the level of expression above 1 fg/µg total RNA.

5-HT₄ receptor isoforms were characterized by RT-PCR in the tumoral adrenal gland and in the five normal adrenal glands. In the tumoral tissue, isoforms c, g, and n could be detected (Table 3). With regard to the normal adrenal glands, isoforms a, c, g, and n were detected in all cases and isoform d was present in three cases, whereas isoforms b, e, f, and h were not detectable in any case.

	Discussion

Top Abstract Introduction Patient and Methods Results Discussion References

Overall, although the cause of Cushing’s syndrome in the case we described remains speculative, it is of interest that in in vitro experiments cisapride greatly augmented ACTH-induced cortisol secretion. In our patient, plasma ACTH was still measurable, as reported in other patients with AIMAH (8), and hypercortisolism might be caused by a combined stimulus on a highly enlarged population of adrenal cells.

The reversal of the clinical picture that we observed in our patient after unilateral adrenalectomy has already been reported in the literature in patients with AIMAH (26). In agreement with the experience of other authors, in our patient the unilateral removal of the largest adrenal gland caused, as above mentioned, a dramatic decrease of cortisol production and improved the clinical picture. Hypocortisolism, which had to be corrected by oral cortisone supplementation, ensued soon after surgery and lasted for several months. At present, plasma and urinary cortisol are normal, and the patient is doing well despite the contralateral enlarged adrenal gland. Therefore, we confirm that unilateral adrenalectomy may be a safe and long-lasting therapy in patients with Cushing’s syndrome due to AIMAH. Whether the remaining adrenal gland will enlarge and cause a recurrence of hypercortisolism in the future is unknown, but until then a condition of permanent Addison’s disease will be avoided.

In conclusion, in the present study we reported the case of a patient with Cushing’s syndrome due to AIMAH, in which cortisol secretion was markedly stimulated by cisapride. Interestingly, the in vivo response was maintained after removal of the largest adrenal gland, also during a state of transient hypocortisolism. In vitro studies confirmed that the response to cisapride was due to the presence of 5-HT₄ receptors and indicated that the expression level was higher than in normal adrenal glands. At present, the role played by illicit receptors in the pathogenesis of Cushing’s syndrome is controversial. Nevertheless, the presence of illicit receptors such as 5-HT₄ receptors in the adrenal glands of patients with hypercortisolism should be investigated, because the eventual availability of specific blockers of 5-HT₄ receptors could be of interest for the potential treatment of such patients.

	Footnotes

 
This work was partially supported by Ministero dell’Istruzione, dell’Università e della Ricerca (Grant 2001062719).

Abbreviations: AIMAH, ACTH-independent macronodular adrenal hyperplasia; AT-1, angiotensin II type 1; AVP arginin-vasopressin; CT, computed tomography; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hCG, human chorionic gonadotropin; 5-HT₄, serotonin 4.

Received December 11, 2002.

Accepted June 16, 2003.

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Top Abstract Introduction Patient and Methods Results Discussion References

 

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