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European Institute for Peptide Research (IFRMP 23), Department of Endocrinology, INSERM, U-413 (H.L., J.M.K.), and Department of Nephrology (M.G.), Centre Hospitalo-Universitaire de Rouen, 76031 Rouen, France; Laboratory of Cellular and Molecular Neuroendocrinology, INSERM, U-413, UA Centre National de la Recherche Scientifique, University of Rouen (H.L., D.C., C.Du., I.L., V.C., C.De., H.V., J.M.K.), 76821 Mont Saint Aignan, France; and Laboratory of Cellular and Molecular Cardiology, INSERM, U-446, Faculty of Pharmacy, University Paris-Sud (R.F.), 92296 Chatenay-Malabry, France
Address all correspondence and requests for reprints to: Dr. Hervé Lefebvre, IFRMP 23, Department of Endocrinology, INSERM, U-413, Hospital of Boisguillaume, Centre Hospitalo-Universitaire de Rouen, 76031 Rouen Cedex, France. E-mail: . herve.lefebvre{at}chu-rouen.fr
Abstract
We have previously shown that serotonin (5-HT) stimulates aldosterone secretion from the human adrenal gland through activation of 5-HT4 receptors. The aim of the present study was to investigate in vivo and in vitro the presence of 5-HT4 receptors in aldosterone-producing adenomas (aldosteronomas). Eight patients with aldosteronoma received a single oral dose of placebo or cisapride (10 mg). Cisapride administration significantly increased plasma aldosterone within 120 min without any significant change in renin, cortisol, or potassium levels. In two patients, a marked decrease in the plasma aldosterone response to cisapride was observed after surgical removal of the tumor. The effects of 5-HT and selective 5-HT4 ligands on aldosterone production from aldosteronoma tissues were studied in vitro using a perifusion system technique. 5-HT and the 5-HT4 receptor agonist cisapride (10-7 M, 20 min) both stimulated aldosterone secretion from aldosteronoma slices. The 5-HT- and cisapride-evoked aldosterone responses were inhibited by concomitant administration of the specific 5-HT4 receptor antagonist GR 113808 (10-7 M, 150 min). PCR amplification revealed the expression of 5-HT4 receptor mRNA in 13 of 14 aldosteronomas studied. Taken together, these data show that most aldosteronomas, like normal glomerulosa cells, express a functional 5-HT4 receptor. Our results also suggest that 5-HT, which can be locally released by intratumoral mast cells, may play a role in the pathophysiology of these tumors.
SEVERAL OBSERVATIONS indicate that serotonin (5-HT) exerts a paracrine stimulation of aldosterone secretion in humans: 1) human adrenocortical extracts contain substantial amounts of 5-HT and its metabolite 5-hydroxyindolacetic acid (1); 2) in the human adrenal cortex, 5-HT is stored in perivascular mast cells, suggesting that 5-HT may be released together with histamine and cytokines during inflammation and allergic processes (1); 3) administration of 5-HT or its precursors, tryptophan and 5-hydroxytryptophan, to healthy volunteers provokes a significant increase in plasma aldosterone levels (PAL) (2, 3, 4); 4) 5-HT stimulates aldosterone production from cultured human zona glomerulosa cells (4, 5); and 5) administration of the mast cell-depleting compound 48/80 to perifused human adrenocortical explants induces a secretory surge of 5-HT, followed by a marked increase in aldosterone secretion (6).
Most biological effects of 5-HT are mediated by a wide variety of seven-transmembrane domain G protein-coupled receptors (for a review, see Ref. 7). Among them, the 5-HT4 receptor subtype is positively coupled to adenylyl cyclase and is selectively activated by benzamide derivatives, such as zacopride and cisapride (7). Alternative splicing of the 5-HT4 receptor transcript has the potential to generate eight receptor isoforms (a–h) (8, 9, 10). There is now clear evidence that the 5-HT4 receptor is expressed by normal human adrenocortical cells (8, 11). In particular, it has been shown in vitro that 5-HT stimulates aldosterone secretion from the human adrenal cortex through activation of 5-HT4 receptors positively coupled to adenylyl cyclase and calcium influx (5, 12, 13). In agreement with these observations, in vivo administration of the 5-HT4 receptor agonists, zacopride and cisapride, to healthy volunteers is followed by a significant increase in PAL (12, 14).
Only a few studies have been conducted to examine the possible action of 5-HT on autonomous adrenal lesions producing aldosterone. A single study has shown that 5-HT stimulates aldosterone secretion in vitro from isolated adrenocortical cells derived from two aldosterone-secreting adenomas (aldosteronomas) (4). It has also been reported that 5-hydroxytryptophan provokes an increase in PAL in patients with idiopathic hyperaldosteronism (IH) (4). Collectively, these observations suggest that hyperplastic and/or adenomatous glomerulosa cells, such as normal glomerulosa tissue, express functional serotonergic receptors.
The aim of the present study was to demonstrate that 5-HT4 receptors are actually expressed in aldosteronomas. The effect of specific 5-HT4 receptor ligands on aldosterone secretion from aldosteronomas was evaluated in vivo and in vitro. Concurrently, the presence of 5-HT4 receptor mRNA in tumor tissue was investigated by RT-PCR and cDNA sequencing.
Materials and Methods
In vivo studies
Patients and treatment. Eight patients (two women and six men; mean age, 48.5 ± 11.9 yr) with newly discovered primary hyperaldosteronism were studied. The experimental protocol was approved by the regional ethics committee. The diagnosis of primary hyperaldosteronism was established on the basis of hypertension, low plasma potassium (mean, 3.2 ± 0.3 mmol/liter), and elevated PAL in recumbency with suppressed renin concentrations in the upright position. The diagnosis of aldosteronoma was established by abdominal computed tomographic scan and unresponsiveness of aldosterone to sodium loading and was further confirmed by histological examination of the tissue after unilateral adrenalectomy that led to normalization of blood pressure and potassium levels. None of the patients had received any therapy that could have influenced the renin-angiotensin-aldosterone system or had undergone surgery before the study. All subjects maintained their regular diet throughout the study. Cisapride [(±)-cis-4-amino-5-chloro-N-(1-[3-(4-fluoro-phenoxy)propyl]3-methoxy-4-piperidinyl)2-methoxybenzamide monohydrate] was purchased from Janssen Pharmaceuticals-Cilag Laboratories (Boulogne-Billancourt, France) as 10-mg tablets, which were subsequently conditioned in capsules. Cisapride and placebo capsules had identical appearance.
Study protocol and hormone assays. Starting at 0900 h, the patients were maintained in a recumbent position for 2 h, and a small catheter was placed into a peripheral vein (time zero) for blood sampling. A single dose of either placebo or cisapride (10 mg) was administered orally in a random simple blind fashion at a 2-d interval. The treatments were administered at 1100 h to avoid fluctuations of ACTH during the study that could have influenced aldosterone secretion. Blood samples for aldosterone, cortisol, renin, and electrolyte measurements were taken at 0, 30, 60, 90, 120, 150, and 180 min. The samples were centrifuged (4 C, 20 min), and the plasma was stored at -20 C until assay. Plasma aldosterone, cortisol, and renin levels were measured in duplicate using, respectively, the following commercial kits: Cis Oris (Gif-sur-Yvette, France), Immunotech (Marseille, France) and ERIA Pasteur (Marne-la-Coquette, France). Intra- and interassay variations were lower than 9% and 11%, respectively, for all assays. Cisapride, at concentrations up to 10-6 M, did not interfere in the assays.
Calculations and statistical analysis.
All data are presented as the mean ± SEM of absolute hormone levels. Two-way ANOVA was performed to detect between-test and time-related differences. Wilcoxon’s test was used after ANOVA for comparisons between and within studies with an 
level of 0.05. For each patient, individual cisapride-induced aldosterone production was evaluated by calculating both the incremental area under the curve (AUC) using the trapezoidal rule between 0–180 min, and the amplitude of the peak of PAL expressed as absolute hormone levels and/or percentage of basal aldosteronemia.
Perifusion experiments
Test substances and reagents. 5-HT was purchased from Sigma (St. Louis, MO). GR 113808 ([1-[2-methylsulphonylamino)ethyl]-4-piperidinyl]methyl] 1-methyl-1H-indole-3-carboxylate, maleate) was provided by Glaxo (Greenford, UK). Cisapride was supplied by Janssen Pharmaceuticals-Cilag Laboratories. DMEM was from Life Technologies, Inc. (Grand Island, NY), and Bio-Gel P2 was from Bio-Rad Laboratories, Inc. (Richmond, CA).
Perifusion technique. Nine different aldosteronomas were studied by use of a perifusion system technique as previously described (1). Among these tumors, three were obtained from patients who had been previously challenged in vivo with cisapride (patients 1, 5, and 8). The remaining six tumors were obtained from patients who had not been tested in vivo. The protocol of collection of the tissue and the experimental procedures were approved by the regional ethics committee. Aldosteronoma tissues were obtained at surgery after informed consent of the patients. For three of the six patients who had not been tested in vivo, a fragment of each tumor was frozen on dry ice and stored at -80 C for RT-PCR experiments (see below), and another fragment was dipped in DMEM for perifusion experiments. Tumor explants were immediately transported to the laboratory in DMEM and transferred into perifusion chambers. The tissue slices 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.
Aldosterone concentrations were determined by RIA as previously described (15). The working range of the assay was 8–2000 pg. None of the test substances interfered in the assay.
RNA extraction and RT-PCR
Expression of 5-HT4 receptors by aldosteronomas was studied by RT-PCR on a series of 14 tumors comprising the 3 aldosteronomas (noted a, b, and c), fragments of which had been used for perifusion experiments, and the aldosteronoma obtained from patient 4 of the in vivo study. The 10 remaining aldosteronomas were provided by a French Endocrinological network for collection of adrenocortical tumors (Réseau COMETE PHRC AOM 95201) and stored at -80 C until RNA extraction. Normal adrenal glands were obtained after informed consent from 2 patients undergoing expanded nephrectomy for kidney cancer. Total RNAs were extracted by a single step procedure according to Chomczynski and Sacchi (16) using Tri-Reagent (Sigma). The concentration and purity of RNAs were determined by spectrophotometry analysis (UV-1605, Shimadzu, Kyoto, Japan). Total RNA (1 µg) from each aldosteronoma and normal adrenal gland was converted into single-stranded cDNA using Superscript II (Life Technologies, Inc., Eragny, France) with oligo(deoxythymidine)12–18 primer (0.5 µg/µl). PCR was performed with the following oligonucleotides designed from the International Patent WO 94/14957 (sequence 7), S1 (5'-CGGGCAGGAGCCTCCTCCGAGAG-3') and AS1 (5'-CAAGGGACAGTCTGGCCCAGAATG-3'), corresponding, respectively, to bases 697–717 and 1020–1043 of the human 5-HT4 receptor cDNA. These two primers hybridize to all 5-HT4 receptor splice variants. 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 (17) (accession no. M17701) were used for semiquantification of reverse transcribed mRNAs. Experiments without RT were carried out using products of total RNA extraction as a substrate for PCR to detect possible amplification of genomic DNA. PCR-based procedures were performed in a final volume of 50 µl containing 10% of the RT mixture, 3 U DNA Taq polymerase (Life Technologies, Inc.), DNA polymerase buffer (Life Technologies, Inc.), 1.5 mM MgCl2, 0.4 mM dNTP, and 20 pmol of each primer. The PCR reactions were performed for 40 cycles (94 C, 40 sec; 62 C, 60 sec; 72 C, 90 sec). The PCR products were analyzed on 1.5% agarose gels, blotted on a nylon membrane, and hybridized with a [32P]ATP-labeled internal oligonucleotide designed on International Patent WO 94/14957 S2 (sequence 7), corresponding to bases 873–893 (5'-CCCTGGGCAGGTGTGGACTGC-3'). Several PCR products were subcloned into pGEM-T (Promega Corp., Charbonnières, France) and sequenced using the Thermo-Sequenase kit (Amersham Pharmacia Biotech, Orsay, France) on a Li-Cor 4200L DNA sequencer (ScienceTec, Les Ulis, France) using fluorescent T7 and T3 primers (MWG-Biotech, Courtaboeuf, France).
Results
In vivo studies
Oral administration of 10 mg cisapride induced a significant increase in PAL (Fig. 1
; P = 0.0026 and F = 10.49, by ANOVA), which reached a maximum at 120 min (P = 0.0016 vs. placebo, using Wilcoxon’s test after ANOVA) and remained elevated during the next 60 min. Stimulation of aldosterone secretion occurred in all patients, with individual incremental AUCs ranging from 53–571 (mean, 229 ± 65) nmol/liter·min (Table 1). The mean absolute value of the secretory peak (2758 ± 526 pmol/liter) was not significantly different from that previously measured in patients with IH (2780 ± 330 pmol/liter) (18). Cisapride treatment did not affect cortisol and potassium levels (data not shown). Renin levels remained suppressed (<2 ng/liter; normal values, 7–19 ng/liter) throughout the study. In patients 1 and 7, the cisapride test was applied again 1 month after removal of the tumor (Fig. 2). In both cases, ablation of the tumor normalized basal PAL and led to a dramatic decrease in the AUC of the cisapride test, from 117 to 6 nmol/liter·min for patient 1 (Fig. 2A) and from 93 to 37 nmol/liter·min for patient 7 (Fig. 2B). For the two patients, the AUCs of the postoperative cisapride tests were, respectively, 23.5 and 3.8 times lower than that previously measured in healthy volunteers (141 ± 35 nmol/liter·min) (14).
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The effect of 5-HT alone or in the presence of the specific 5-HT4 receptor antagonist GR 113808 on aldosterone production was studied on two perifused aldosteronomas. Typical profiles illustrating the kinetics of the aldosterone response to 5-HT are shown in Fig. 3. 5-HT (10-7 M, 20 min) induced a transient stimulation of aldosterone secretion, reaching a maximum of +313% 40 min after the onset of 5-HT infusion (Fig. 3A). During prolonged administration of GR 113808 (10-7 M, 150 min), the stimulatory effect of 5-HT on aldosterone production was reduced by 66% (Fig. 3B). The effect of the 5-HT4 receptor agonist cisapride on aldosterone secretion from seven additional aldosteronomas, including the tumors obtained from patients 1, 5, and 8, was examined using a series of experiments similar to that presented in Fig. 3. In all tumors cisapride (10-7 M, 20 min) induced a marked and sustained increase in aldosterone production. The mean amplitude of the aldosterone response was 178 ± 57% of the basal level, ranging from 82–447%. The stimulatory effect of cisapride was totally abolished by prolonged administration of GR 113808 (10-7 M, 150 min). A representative profile illustrating the effect of cisapride alone and during prolonged administration of GR 113808 on aldosterone secretion by perifused aldosteronoma slices is presented in Fig. 4, A and B.
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The occurrence of 5-HT4 receptor mRNA was investigated by RT-PCR amplification in aldosteronoma tissues and in 2 normal human adrenal glands using specific primers hybridizing to all 5-HT4 receptor splice variants. 5-HT4 receptor PCR products were detected in the 3 aldosteronomas that had been shown to respond in vitro to 5-HT and/or the 5-HT4 receptor agonist cisapride (a–c, Fig. 5A), in the aldosteronoma removed from patient 4 that had been shown to respond in vivo to cisapride treatment (p4, Fig. 5B), in 9 of the retrospective series of 10 aldosteronomas (d–m, Fig. 5C), and in 2 normal adrenal glands (ag1 and ag2, Fig. 5D). In contrast, no hybridization signal was detected in experiments without RT. Individual bands obtained from aldosteronoma 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).
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We have previously reported that in the normal adrenal gland 5-HT exerts a stimulatory effect on aldosterone secretion in vitro through activation of a 5-HT4 receptor subtype (5, 12). We have subsequently shown that the 5-HT4 receptor agonists, zacopride and cisapride, induce a substantial increase in PAL in healthy volunteers and patients with IH (12, 14, 18). In the present study we show that oral administration of a single dose of 10 mg cisapride to patients with aldosteronoma provokes a significant increase in PAL. The maximum response was not statistically different from that previously observed in patients with idiopathic hyperaldosteronism (18), indicating that a cisapride stimulatory test would not have any interest for the clinical discrimination between IH and aldosteronomas. The stimulatory effect of cisapride on aldosterone secretion is consistent with the known effects of serotonergic ligands in patients with aldosteronoma. Administration of the weak histaminergic and 5-HT2C receptor antagonist cyproheptadine had no influence on PAL (19), whereas ketanserin, which is a potent 5-HT2A/2C receptor antagonist (7), induced a decrease in both aldosterone and cortisol levels, suggesting an indirect action on aldosteronoma tissue through inhibition of ACTH secretion (20). This hypothesis is strongly supported by the following observations: 1) ketanserin is devoid of significant affinity for the 5-HT4 receptor (21); 2) 5-HT is able to stimulate ACTH secretion through activation of 5-HT2A/2C receptors (22); and 3) aldosteronomas are highly sensitive to the action of ACTH (23). In the present study the lack of effect of cisapride treatment on cortisol and potassium levels as well as the dramatic decrease in the aldosterone response to cisapride observed in two patients after surgical removal of the tumor indicate that the preoperative cisapride-evoked increase in PAL could be ascribed to direct stimulation of aldosteronoma tissue by the 5-HT4 receptor agonist. Interestingly, the amplitude of the postoperative aldosterone response to cisapride was much lower than that previously described in healthy volunteers, suggesting that the responsiveness of normal glomerulosa cells to 5-HT had been profoundly depressed by the prolonged state of preoperative autonomous hyperaldosteronism. In agreement with this observation, it has been shown that aldosterone response to angiotensin II is transiently suppressed in the normal adrenal gland after surgical removal of aldosteronoma (24).
In addition to the clinical studies, pharmacological and molecular experiments were conducted to characterize 5-HT4 receptors in aldosteronoma tissue. We found that 5-HT and the 5-HT4 receptor agonist cisapride were able to stimulate aldosterone production by aldosteronoma slices. The observation that the 5-HT4 receptor antagonist GR 113808 inhibited the secretory response of aldosteronoma explants to both 5-HT and cisapride provided evidence that the stimulatory effect of 5-HT on aldosterone secretion from adenomatous glomerulosa cells was mediated through a 5-HT4 receptor subtype. To further explore this issue, we investigated the expression of the 5-HT4 receptor by aldosteronoma tissues using RT-PCR amplification of 5-HT4 receptor mRNAs with primers hybridizing to the sequence common to all 5-HT4 receptor splice variants. PCR products were detected in the tumor removed from patient 4, whose aldosteronoma responded in vivo to cisapride, and in the 3 aldosteronomas whose secretory activity could be stimulated by 5-HT and/or cisapride in vitro. Specific PCR products were also detected in 9 of a retrospective series of 10 aldosteronomas as well as in 2 normal adrenal glands. The fact that numerous mast cells, which are the major source of 5-HT in the normal adrenal gland, are found in aldosteronoma tissues (25) suggests that 5-HT released by intratumoral mast cells may exert a tonic stimulatory effect on aldosterone secretion and thus may play a significant role in the pathophysiology of these tumors.
Taken together, the present results demonstrate that most aldosteronomas, like normal glomerulosa cells, express a functional 5-HT4 receptor. These data suggest that selective 5-HT4 receptor antagonists may represent a new approach in the treatment of primary hyperaldosteronism.
Acknowledgments
We thank Drs. P. Grise, D. Pavard, and O. Rousseau, who kindly provided human adrenal tissue, and M. Guervin for technical assistance.
Footnotes
This work was supported by the Conseil Régional de Haute-Normandie, IFRMP 23, INSERM, U-413, the CHU de Rouen, and the Réseau COMETE (PHRC AOM 95201).
Abbreviations: AUC, Area under the curve; 5-HT, serotonin; IH, idiopathic hyperaldosteronism; PAL, plasma aldosterone levels.
Received August 16, 2001.
Accepted November 30, 2001.
References
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) or placebo (
) was administered orally at time zero (arrow). *, P < 0.05; **, P < 0.02.
