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Original Article |
European Institute for Peptide Research (Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23), Laboratory of Cellular and Molecular Neuroendocrinology (D.C., I.L., J.-M.K., H.V., H.L.), Institut National de la Santé et de la Recherche Médicale (INSERM) U413, UA Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France; Laboratory of Oncologic Genetics (F.P., C.B.), Centre Henri Becquerel, 76038 Rouen, France; Department of Endocrinology (J.B.), Centre Hospitalier Universitaire (CHU) Cochin, 75031 Paris, France; Department of Endocrinology (P.C.), CHU of Toulouse, 31403 Toulouse, France; Department of Endocrinology (H.L., J.-M.K.), INSERM U413, CHU of Rouen, 76031 Rouen, France; Division of Endocrinology (A.L.), Department of Medicine, Research Center Hôtel-Dieu, CHUM, Montréal, Canada H2W 1T8; Department of Endocrinology (A.T.), CHU of Bordeaux, 33604 Bordeaux, France; and Department of Endocrinology (J.Y.), CHU Kremlin-Bicêtre, 94275 Le Kremlin Bicêtre, France
Address all correspondence and requests for reprints to: Dr. Hervé Lefebvre, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23, Department of Endocrinology, Institut National de la Santé et de la Recherche Médicale U413, 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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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Several observations indicate that cortisol production is controlled by aberrant membrane receptors in both adrenal tumors and ACTH-independent bilateral macronodular adrenal hyperplasias (AIMAH) causing Cushing’s syndrome (11). Abnormal responses of cortisol secretion to administration of ligands for eutopic receptors have also been described in patients with primary adrenal hypercortisolism (11). In particular, cisapride and metoclopramide have been shown to stimulate cortisol secretion in patients with AIMAH and subclinical or patent Cushing’s syndrome, suggesting an overexpression of 5-HT4 receptors in this type of lesion (11, 12). However, the mechanisms involved in the hyper-responsiveness of the adrenal hyperplastic tissue to 5-HT4 receptor agonists remain currently unknown.
The aim of the present study was to investigate quantitatively and qualitatively the expression of the 5-HT4 receptor in both normal adrenal cortex and tissues removed from six patients with cisapride-responsive AIMAH causing subclinical or patent Cushing’s syndrome. Quantitative PCR was performed to compare the levels of expression of the 5-HT4 receptor in hyperplastic adrenal tissues with those of normal adrenal cortices. In addition, the 5-HT4 receptor variants expressed by the tissues were identified by use of RT-PCR and subsequently subcloned and sequenced.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Six patients with AIMAH causing subclinical or overt Cushing’s syndrome were studied. Patient 1, a 73-yr-old woman, was referred for bilateral adrenal hyperplasia discovered incidentally by abdominal ultrasound. She had experienced hypertension for 8 yr. Clinical examination showed typical signs of hypercortisolism including facial plethora, epidermal atrophy, and proximal myopathy. Abdominal magnetic resonance imaging revealed a nodular hyperplasia of the two adrenal glands reaching a diameter of 3.5 cm on the right and 2.0 cm on the left. The clinical presentation of patient 2, a 63-yr-old woman, has been previously described (13). Briefly, she presented with a 12-month history of hypertension, proximal-muscle weakness, decrease in memory, and weight gain of 19 kg during the last 9 yr. She had four full-term pregnancies, at which time she had a Cushingoid distribution of fat and weight gain of 18–22 kg. Postpartum, her weight had rapidly returned to baseline. She had undergone hysterectomy and bilateral oophorectomy at 61 yr of age because of uterine prolapse. At examination, she had central obesity, mild facial plethora and hirsutism, supraclavicular fat pads, and proximal muscle weakness. An abdominal computerized tomography (CT) scan showed bilateral macronodular adrenal hyperplasia with nodules measuring up to 4 x 3.5 cm on the right and 2.5 x 4 cm on the left. Patient 3, a 50-yr-old woman, was admitted for bilateral adrenal tumors incidentally discovered during abdominal CT scan performed for lumbar pain. She had no history of hypertension. Her menses had stopped 6 months previously. Clinical examination revealed no sign of hypercortisolism. On abdominal CT scan, the adrenal nodules reached 5 x 3 cm on the right and 3 x 2 cm on the left. An iodocholesterol scan performed without dexamethasone suppression showed bilateral adrenal uptake of the tracer predominant on the right gland. Patient 4, a 46-yr-old man, presented with a history of weight gain, muscle weakness, and hypertension. Abdominal CT scan revealed enlarged adrenal glands with nodules measuring up to 2.5 cm on the right and 3.5 cm on the left. Patient 5 was a 34-yr-old woman referred for central obesity, hypertension, and diabetes mellitus. On abdominal CT scan, adrenal glands were hyperplastic with nodules measuring up to 2 cm on the left and 5 cm on the right. Patient 6, a 74-yr-old man, was referred for symptoms suggestive of hypercortisolism comprising facial plethora and erythrosis, easy bruisability, and hypertension. Abdominal CT scan showed multiple adrenal nodules reaching 5 cm on the left and 3 cm on the right.
The results of the initial endocrine investigations for the six patients are summarized in Table 1
. Potentially aberrant membrane hormone receptors were systematically searched for, after informed consent of the patients, by using a clinical protocol previously described (14). The study was approved by the regional and/or institutional ethics committees. Briefly, plasma cortisol levels were measured in response to a posture test, a standard mixed meal, administration of 250 µg ACTH1–24 iv used as a reference test, 100 µg GnRH iv, 100 µg TRH iv, 1 mg glucagon iv, 1 mg terlipressin [a precursor of lysine vasopressin (LVP), Glypressine, Ferring Pharmaceuticals Ltd., Gentilly, France], or 10 IU arginine vasopressin im. In addition, all patients underwent a cisapride stimulation test consisting of an oral administration of 10 mg cisapride (Prepulsid, Laboratoires Janssen-Cilag, Boulogne-Billancourt, France) and plasma cortisol measurements at 0, 60, and 120 min. In patient 2, the adrenal sensitivity to 5-HT4 receptor agonists was also investigated with 10 mg metoclopramide orally (Maxeran, Aventis Pharma Inc., Laval, Québec, Canada). Plasma cortisol was measured by using commercial immunofluorometric kits and ACTH by immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA). For all tests, a positive cortisol response was arbitrarily defined as a 25% increase in plasma cortisol. The tests inducing a positive plasma cortisol response are reported for the six patients in Table 1. In all cases, cisapride induced a significant increase in plasma cortisol levels. In addition, plasma ACTH levels remained undetectable throughout the study in all patients (data not shown).
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All patients with AIMAH underwent bilateral adrenalectomy, except patient 2 in which a biopsy of the left adrenal gland was obtained. Fragments of the tissue nodules were immediately stored at -80 C until RNA extraction. Normal adrenal glands were obtained from seven patients undergoing expanded nephrectomy for kidney cancer. The protocol of collection of the tissues and the experimental procedures were approved by the regional and/or institutional ethics committees, and written informed consent was obtained from all subjects.
Amplification of the 5-HT4 receptor by RT-PCR.
Total RNA from the six hyperplasias and seven normal adrenal glands was extracted by the acid guanidium-thiocyanate-phenol-chloroform procedure by using Tri Reagent (Sigma, St. Louis, MO). The concentration of total RNA was determined by measuring the optical density at 260 nm. Total RNA (1 µg) from each tissue was converted to single stranded cDNA using SuperScript II from Life Technologies, Inc. (Eragny, France) with oligo (dT)12–18 primer (0.5 µg/ml). Amplification of the domain common to all 5-HT4 receptor isoforms was performed by PCR with the primers S1 and As1 (Table 2). S1 and As1 primers were designed in exon 4 [nucleotides (nts) 697–717] and exon 5 (nts 1020–1043), respectively, of the human 5-HT4 receptor gene (Fig. 1). Two other primers (5'-TGCTGAGTAYGTCGTGGAGTC-3' and 5'-TTGGTGGTGCAGGAKGCATTGC-3'), corresponding to bases 297–317 and 467–488, respectively, of the cloned glyceraldehyde-3-phosphate dehydrogenase sequence (GenBank accession no. M17701) were used for semiquantitation of reverse transcribed mRNAs. The PCRs were performed for 40 cycles (94 C, 40 sec; 50 C, 60 sec; 72 C, 90 sec). The amplified products were analyzed in 1.5% agarose gels, blotted on a nylon membrane, and hybridized with a [32P]ATP-labeled internal oligonucleotide S2 (Table 2, nts 873–893). In addition, PCR products were subcloned into pGEM-T (Promega Corp., Charbonnières, France) and sequenced using the Thermosequenase kit (Amersham, Orsay, France) on a Li-Cor 4200L DNA sequencer (ScienceTec, Les Ulis, France) using fluorescent T7 and T3 primers (MWG-Biotech, Courtaboeuf, France).
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). Briefly, 1 µg of total RNA was reverse transcribed in triplicate using Maloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) and oligo dT primer as described above. The cDNA prepared was diluted and aliquoted into microtiter plates. For each 25-µl TaqMan reaction, 5 µl cDNA was mixed with 1 µl water, 12.5 µl TaqMan Universal PCR Master Mix 2X (Applied Biosystem, Courtaboeuf, France), 2 µl sense primer (2 µM), 2 µl antisense primer (2 µM), and 2.5 µl TaqMan probe (2 µM). PCR parameters were 50 C for 2 min, 95 C for 10 min, 40 cycles of 95 C for 15 sec and 60 C for 1 min. In addition, parallel assays using the same cDNA pools were performed using primers and probe to the housekeeping gene porphobilinogen deaminase (PBGD; Table 3). Quantitative RT-PCR was performed using an ABI Prism 7700 sequence detector system (Applied Biosystem) and analyzed using relative expression to PBGD, as previously described (15). Briefly, the level of expression in each sample was normalized by dividing copies per nanogram total RNA of 5-HT4 receptor gene by copies per nanogram total RNA of the housekeeping gene PBGD, and expressed as a percentage. This mode of calculation allows to correct for both RNA quality and quantity. In each group of tissues, i.e. normal and hyperplastic glands, data are presented as mean ± SEM.
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Total RNA was extracted and reverse transcribed as described above. Amplification of the cDNAs encoding the different 5-HT4 receptor C-terminal splice variants was performed by PCR using primer S1 (Table 2
), which hybridizes to all 5-HT4 receptor messages and splice variant-specific reverse primers (Table 2 and Fig. 1). The 5-HT4(h) variant was amplified using the forward primer Fwh, which is specific for cDNAs containing the 5-HT4(h) exon, and the reverse primer Revh, which hybridizes to all 5-HT4 receptor messages. All PCR-based procedures were performed in a final volume of 50 µl containing 10% of RT mixture, 3 U DNA Taq Polymerase (Life Technologies, Inc.), DNA Polymerase buffer (Life Technologies, Inc.), 1.5 mM MgCl2, 0.4 mM deoxynucleoside triphosphate, and 20 pmol of each primer. The PCRs were performed for 40 cycles (94 C, 40 sec; 50 C, 60 sec; 72 C, 90 sec). For each tissue, the PCR products were analyzed in 1.5% agarose gels, blotted on a nylon membrane, and hybridized with the [32P]ATP-labeled oligonucleotide S2. All PCR products were subcloned and sequenced as described above. In addition, the full-length cDNAs encoding the isoforms expressed by hyperplasias P2 and P5 were amplified by PCR using the primer exon 1 (5'-ATGGACAAACTTCATGCTAATGTG-3') corresponding to the first 24 nucleotides of the cloned 5-HT4 receptor sequence (GenBank accession no. Y10437) and splice variant specific reverse primers (Table 2 and Fig. 1). PCR products of the expected sizes were subcloned into pGEM-T and sequenced. For each 5-HT4 receptor splice variant, the full-length cDNA sequence was determined from six clones.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The occurrence of 5-HT4 receptor mRNA in adrenocortical hyperplasias and normal adrenal cortex was investigated by RT-PCR amplification using oligonucleotides hybridizing to all receptor variants. 5-HT4 receptor PCR products were detected in all of the tissues studied (Fig. 2). Individual bands obtained from hyperplasia and normal adrenal tissue reverse-transcribed RNAs were excised, ligated into pGEM-T, and sequenced. All sequences corresponded to the published sequence of the 5-HT4 receptor cDNA (data not shown). The quantitative expression of 5-HT4 receptor mRNA by the tissues was then determined by TaqMan PCR. When expressed as arbitrary units normalized to PBGD, 5-HT4 receptor expression ranged from 1.55% to 33.9% (mean, 13.1% ± 5.39%) in hyperplasias vs. 0.48% to 3.72% (mean, 2.13% ± 0.46%) in normal adrenal glands (Fig. 3). Two hyperplasias (P2 and P5) had levels of 5-HT4 receptor expression similar to those of normal tissues, i.e. 1.65% and 1.55%, respectively. In the four remaining hyperplasias, 5-HT4 receptor expression was 3 to 16 times higher than in normal glands (Fig. 3).
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5-HT4 receptor isoforms were characterized by RT-PCR amplification in both hyperplastic adrenal cortex and normal adrenal glands. In the two types of tissues, the profile of expression of 5-HT4 receptor variants was variable from one sample to the other (Table 4). Hyperplastic tissues were found to express isoforms a–d, f, h, and n. Five hyperplasias expressed isoform a, four expressed isoform n, three expressed isoform b, two expressed isoform d, and one expressed isoform h. Isoforms c and f were expressed by all hyperplasias, whereas isoforms e and g were not detected in any hyperplastic tissue. The 5-HT4 receptor variants present in hyperplasias P2 and P5, which did not overexpress the 5-HT4 receptor, i.e. variants a–d and f, could also be observed in normal adrenal glands (Fig. 4). Isoform n was detected in all normal adrenal tissues, whereas isoforms a, c, and f were characterized in six of the seven glands studied. In addition, five adrenal glands expressed isoform b, and one expressed isoform d. None of the normal adrenal tissues expressed isoforms e, g, and h. Finally, the cDNAs encoding isoforms a–d and f in hyperplasias P2 and P5 were subcloned and full-length sequenced to detect a possible mutation causing gain-of-function of the 5-HT4 receptor. All sequences were identical to those of the coding regions of the published 5-HT4 receptor gene (data not shown).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In two hyperplasias (P2 and P5), the level of 5-HT4 receptor expression was similar to that of normal adrenal glands. Owing to the fact that 5-HT4 receptor variants may exhibit different coupling efficiencies (21), we thus hypothesized that, in some cases, the in vivo responsiveness of hyperplastic adrenal glands to cisapride and/or metoclopramide could be ascribed to expression of 5-HT4 receptor isoforms that differ from those present in the normal adrenal cortex. We found that the 5-HT4 receptor variants expressed by hyperplasias P2 and P5, i.e. variants a–d and f, could also be detected in normal adrenocortical tissue, indicating that the pathological in vivo cortisol response to cisapride was not attributable to the presence of ectopic 5-HT4 receptor isoforms in these two tissues. Isoform h was characterized in one hyperplasia overexpressing the 5-HT4 receptor (P6) but not in the series of normal adrenal tissues. However, several observations indicate that the enhanced responsiveness of the hyperplastic tissue to cisapride could not be accounted for by aberrant expression of this isoform: 1) the potencies and efficacies of cisapride to stimulate cAMP formation in COS-7 cells transfected with either the human 5-HT4(h) isoform or the C-terminal splice variants, i.e. the 5-HT4(a) and 5-HT4(b) isoforms, are similar (5); 2) a study of 5-HT4(h) mRNA distribution in human tissues has shown that isoform h can also be expressed in the normal adrenal gland (5). Recent studies have shown that G protein-coupled receptors can form homodimers and/or heterodimers that modulate receptor function (22, 23). It is therefore possible that differences in the expression profiles of 5-HT4 receptor splice variants could induce a functional variability in 5-HT4 receptor-mediated tissue responses. In particular, an abnormal expression profile of 5-HT4 receptor isoforms could be involved in the pathological cortisol response to cisapride in some patients with AIMAH. Consistent with this hypothesis, the expression profiles of the 5-HT4 receptor splice variants in hyperplasias 2 and 5, i.e. a-/b+/c+/d+/e-/f+/g-/h-/n- and a+/b+/c+/d+/e-/f+/g-/h-/n-, respectively, were not observed in the series of normal adrenal cortices.
Gain-of-function mutations of G protein-coupled receptors leading to increased cAMP production and cellular proliferation have been described in various endocrine diseases, including familial neonatal hyperthyroidism and testotoxicosis (24, 25). In addition, site-directed mutagenesis studies have shown that mutations occurring within the third intracellular loop of the 5-HT4 receptor, such as mutation A258L, increase the constitutive activity of the receptor as well as its affinity for 5-HT and its coupling efficiency (16). We have thus examined whether the enhanced sensitivity of the hyperplastic adrenal tissues P2 and P5 to 5-HT4 receptor agonists could be ascribed to the occurrence of somatic point mutations causing gain of function of 5-HT4 receptors within the adrenal tissue. Our data revealed that, in the hyperplastic adrenal glands removed from patients 2 and 5, the sequences of the cDNAs encoding the isoforms expressed by the tissues (a–d and f) were identical to those of the coding regions of the published human 5-HT4 receptor gene. This observation indicates that the cellular defect leading to enhanced adrenal sensitivity to 5-HT and hyperplasia cannot be accounted for by an activating mutation of the 5-HT4 receptor.
In conclusion, our results show for the first time an overexpression of the eutopic 5-HT4 receptor in AIMAHs responsive in vivo to cisapride administration. In some cases however, the level of expression of the receptor is similar to that of normal adrenal tissue. In this situation, the enhanced sensitivity of the tissue to cisapride is not due to ectopic expression of 5-HT4 receptor isoforms or to the occurrence of somatic gain-of-function mutation of the receptor.
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Acknowledgments |
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Footnotes |
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Abbreviations: AIMAH, ACTH-independent bilateral macronodular adrenal hyperplasias; CT, computerized tomography; 5-HT, serotonin; LVP, lysine vasopressin; nts, nucleotides; PBGD, porphobilinogen deaminase.
Received July 17, 2002.
Accepted September 17, 2002.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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