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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-1453
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 4 1566-1572
Copyright © 2006 by The Endocrine Society

Evidence for a Role of Vasopressin in the Control of Aldosterone Secretion in Primary Aldosteronism: in Vitro and in Vivo Studies

Véronique Perraudin, Catherine Delarue, Hervé Lefebvre, Jean-Luc Do Rego, Hubert Vaudry and Jean-Marc Kuhn

Institut National de la Santé et de la Recherche Médicale Unité 413 (V.P., C.D., H.L., J.-L.D.R., H.V., J.-M.K.), Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (IFRMP 23), University of Rouen, 76821 Mont-Saint-Aignan, France; and Institut National de la Santé et de la Recherche Médicale Center for Clinical Investigation 204 (H.L., J.-M.K.), University Hospital of Rouen, 76031 Rouen, France

Address all correspondence and requests for reprints to: Dr. Hubert Vaudry, Institut National de la Santé et de la Recherche Médicale Unité 413, Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (Institut Fédératif de Recherche Multidisciplinaires sur les Peptides 23), University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry{at}univ-rouen.fr.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Arginine vasopressin (AVP) stimulates steroid secretion from the normal human adrenal gland and some cortisol-producing adrenocortical tumors or hyperplasia through activation of the V1a receptor.

Objective: The objective of the study was to investigate in vitro and in vivo the possible involvement of AVP in the physiopathology of primary aldosteronism.

Design: The design of the study included immunohistochemical, pharmacological, and molecular studies on aldosterone-producing adenoma (APA), followed by a monocentric, crossover trial of the orally active V1a receptor antagonist, SR 49059, in a double blind, randomized, and placebo-controlled fashion.

Setting: The study was conducted at a university hospital and research laboratory.

Patients: The study population included eight untreated patients with primary aldosteronism, four with APA and four with idiopathic hyperaldosteronism.

Main Outcome Measures: Aldosterone secretion of APA cells in vitro and plasma aldosterone, renin, and ACTH were measured.

Intervention: SR 49059 (200 mg once daily) or placebo was administered during two 1-wk treatment periods separated by a 2-wk washout.

Results: We observed the occurrence of AVP-containing cells in APA tissues. Administration of AVP to perifused APA cells induced an increase in aldosterone production, which was inhibited by a specific V1a antagonist. RT-PCR analysis showed the expression of V1a receptor mRNA in most APAs studied. In APA patients, SR 49059 did not induce any effect on basal aldosterone secretion but provoked a plasma aldosterone response to orthostatism (P < 0.03) and strengthened the positive correlation between plasma aldosterone and ACTH.

Conclusions: The present study indicates that functional V1a receptors are present in APA and suggests that AVP may exert an autocrine/paracrine control of aldosterone secretion in APA tissues.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
THE SECRETORY ACTIVITY of the adrenal gland is primarily regulated by corticotropic hormones (mainly ACTH and angiotensin II) and circulating concentrations of K+. However, there is now increasing evidence that, besides humoral factors, several locally produced neurotransmitters and neuropeptides including arginine vasopressin (AVP) can participate in the regulation of steroidogenesis (1, 2, 3). In fact, many studies carried out in various animal species have clearly demonstrated that AVP, produced within the adrenal gland, can stimulate corticosteroid secretion through a paracrine mechanism (4, 5, 6, 7). In addition, we previously reported the presence of AVP-containing cells in the human adrenal gland in both the cortex and the medulla, and we have shown that AVP enhances in vitro cortisol secretion from the normal human adrenal cortex (8). Similarly, a direct stimulatory effect of AVP on aldosterone production has also been demonstrated in man (9).

AVP exerts its physiological effects through three different types of receptors, the vascular V1a (or V1) and the pituitary V1b (or V3) receptors, which both activate phospholipase C (10, 11, 12), and the renal V2 receptor positively coupled to adenylyl cyclase (13, 14). The AVP-induced stimulation of human adrenocortical cells can be accounted for by activation of V1a receptors (8, 9). It thus appears that AVP may physiologically regulate corticosteroid secretion in the human adrenal gland. Recent data indicate that AVP may also be involved in the physiopathology of adrenal Cushing’s syndrome. In particular, we previously reported a case of cortisol-producing adenoma expressing eutopic V1a receptors that exhibited exaggerated response to AVP (15). Since this initial observation, additional cases of AVP-dependent cortisol-secreting adrenocortical hyperplasia and tumors have been described by several groups (16, 17, 18). However, the sensitivity of aldosterone-producing adenomas (APAs) to AVP has never been evaluated.

In the present study, we investigated in vitro and in vivo the possible role of AVP in the control of aldosterone secretion in patients with primary aldosteronism (PA). The presence of AVP in APA tissues has been explored by immunohistochemistry. We also examined in vitro the effect of AVP on aldosterone secretion by perifused APA slices and the expression of the V1a receptor in APA by RT-PCR analysis. Finally, we studied in vivo the effect of the selective nonpeptidic V1a receptor antagonist SR 49059 on plasma aldosterone levels in patients with PA including APA and idiopathic hyperaldosteronism (IH).


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
In vitro studies

Tissues. APAs were obtained at surgery, after informed consent, from patients presenting typical signs and symptoms of PA. Eighteen adenomas were provided by a French network for collection of adrenocortical tumors (Réseau COMETE, Cortico et Medullo: Etude des Tumeurs Endocrines, Programme Hospitalier de Recherche Clinique AOM 95201). Tumor tissues were either collected and frozen at –80 C for RNA extraction or immersed in DMEM (GIBCO, Grand Island, NY) supplemented with 0.5% antibiotic-antimycotic solution and rapidly transported to the laboratory for perifusion experiments. The protocol of collection of the tissue and the experimental procedures were approved by the regional ethics committee (CCPPRB-HN, Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de Haute-Normandie, Loi Huriet, January 1990).

Immunohistological procedure. The tumor tissues obtained at surgery were immediately fixed in 10% buffered formalin (pH 7.4) and embedded in paraffin, or frozen and stored at –80 C. Paraffin-embedded tissues were subsequently cut into 7-µm sections and processed for immunoenzymatic labeling. Frozen tumor explants were immersed in embedding medium (O.C.T. Tissue Tek; Reichert Jung, Nussloch, Germany), sliced into 7-µm sections with a cryostat (Frigocut 2700, Reichert Jung) and processed for immunofluorescence labeling.

For immunoenzymatic labeling, tissue sections were deparaffinized in cyclohexane and hydrated through a graded series of ethanol to water. Endogenous peroxidase activity was blocked with 3% H2O2 at room temperature for 5 min, and nonspecific labeling was reduced by incubating the slices with 5% normal goat serum for 30 min. Tissue sections were incubated with the monoclonal AVP antibody diluted 1:1000 (19) in a humid atmosphere, at 4 C, overnight. After rinsing in PBS, the bound AVP antibody was visualized by streptavidin-biotin-peroxidase with the goat Immunocruz staining system (Santa Cruz Biotechnology, La Jolla, CA) according to the manufacturer’s instructions, using 3,3'-diaminobenzidine as the color-developing reagent. Slices were counterstained with hematoxylin, dehydrated through a graded series of ethanol and cyclohexane washes, and mounted in Eukitt (Kindler GmbH, Freiburg, Germany). The preparations were examined under an Eclipse E-600 microscope (Nikon, les Ulis, France) equipped with a CCD DXC950 camera (Sony, Paris, France).

For double-immunofluorescence labeling, tumor slices were incubated simultaneously with two primary antibodies, i.e. the monoclonal anti-AVP antibody diluted 1:1000 and a rabbit antiserum (code no. 333-1506) directed against peptide WE14 whose sequence corresponds to the amino acid sequence 324–337 of human chromogranin A diluted 1:500 in PBS containing 0.3% Triton X-100 and 1% BSA (Roche Diagnostics, Mannheim, Germany). The sections were rinsed in four different baths of PBS (10 min each) and incubated at room temperature for 90 min with two secondary antibodies, i.e. GAM/Alexa-488 (Alexa-488-conjugated goat antimouse {gamma}-globulins; Molecular Probes, Leiden, The Netherlands) diluted 1:100 and GAR/Alexa-568 (Alexa-568-conjugated goat antirabbit {gamma}-globulins, Molecular Probes) diluted 1:500. The sections were rinsed in PBS for 1 h and dipped into Bouin’s fixative (picric acid 71%, formaldehyde 9.6%, acetic acid 0.05% in PBS) at 4 C for 30 min. Finally, slices were rinsed, mounted in PBS-glycerol (1:1), coverslipped, and examined using a confocal laser-scanning microscope (Leica, Heidelberg, Germany) equipped with a Diaplan optical system and an argon/krypton ion laser (excitation wavelengths: 488/568/647 nm).

To verify the specificity of the immunoreaction, the following controls were performed: substitution of the AVP antibody with PBS and incubation of the AVP antibody preabsorbed with synthetic AVP (10–5 M).

Perifusion experiments. The tumor tissue was diced into small pieces (1–2 mm3), rinsed with fresh DMEM, mixed with Bio-Gel P2 (Bio-Rad, Ivry-sur-Seine, France), and transferred into polystyrene columns as previously described (8). The tissue was perifused with DMEM, continuously gassed with 95% O2-5% CO2 mixture, at a constant flow rate (260 µl/min). The pH (7.4) and temperature (37 C) were kept constant throughout the experiment. The tumor slices were allowed to stabilize for 2 h, and then test substances, dissolved in DMEM, were infused at the same flow rate as DMEM alone. Effluent fractions were collected at 5-min intervals and kept at –20 C until assay. Synthetic AVP and [d(CH2)5,Tyr(OMe)2]AVP were purchased from Sigma Chemical (St. Louis, MO).

Aldosterone concentrations were determined in the perifusion fractions by RIA as previously described (20). The working range of the assay was 8–2000 pg. The specificity of the antibodies was such that the assay could be carried out directly on the effluent perfusate without any extraction or purification procedure (21).

The perifusion profile represents the secretion pattern of two experiments. The concentration of aldosterone released in the effluent perfusate was expressed as a percentage of the basal values calculated as the mean of eight samples (40 min) taken just before administration of the test substances.

RNA extraction and RT-PCR analysis. Total RNA from adrenal tumor was extracted by a single-step procedure according to Chomczynski and Sacchi (22) using the Tri reagent (Sigma). The concentration and purity of RNA were determined by spectrophotometry (UV-1605; Shimadzu, Kyoto, Japan). RT-PCR was performed according to standard protocols. Briefly, single-stranded cDNA was generated by reverse transcription (RT) of 5 µg total RNA. RT was conducted in a 20-µl final reaction containing 25 mM Tris-HCl (pH 8.3), 37.5 mM KCl, 1.5 mM MgCl2, 50 mM dithiothreitol, 25 µg/ml oligodT15 (Promega, Charbonnières, France), and 200 U Maloney murine leukemia virus reverse transcriptase. Two sets of specific oligodeoxynucleotides were designed: for the V1a receptor, 5'-CGGCTTCATCTGCTACAACATC-3' and 5'-CGAGTCCTTCCACATACCCGT-3', corresponding, respectively, to bases 731–752 and 1215–1235 of the human V1a receptor cDNA (accession no. BC074804) and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-ATCCCATCACCATCTTCCAG-3' and 5'-AGGGATGATGTTCTGGAGAGC-3', corresponding respectively to bases 317–336 and 706–726 of the human GAPDH sequence (accession no. NM 002046). To ensure cDNA-specific amplification, primers for the V1a receptor were chosen flanking an intron. PCR was carried out in a reaction volume of 50 µl containing 1:10 RT reaction and primers (10 pmol) in 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 2.5 mM MgCl2, 0.1% Triton X-100, 0.2 mM each deoxynucleotide triphosphate, and 2.5 U Taq polymerase (Life Technologies, Eragny, France). After a 2-min denaturation at 94 C, 35 cycles of amplification were performed (30 sec denaturation at 94 C, 60 sec annealing at 55 C, and 90 sec extension at 72 C) in a DNA Thermal Cycler (PerkinElmer, Courtaboeuf, France). PCR products were subcloned into p GEM-T (Promega) and sequenced to verify the specificity of the amplified products.

In vivo studies

This study was a monocentric, repeated oral dose, double blind, randomized, placebo-controlled, crossover trial of SR 49059 given once daily for 7 d.

Subjects and treatment. Eight patients (four men and four women; mean age 48.4 yr; mean weight 78.3 kg) with PA were studied. The experimental protocol was approved by the CCPPRB-HN (Comite Consultatif de Protection des Personnes dans la Recherche Biomédicale de Haute-Normandie). The diagnosis of PA was established on the basis of hypertension, low plasma potassium (mean 3.04 ± 0.20 mmol/liter), elevated plasma aldosterone level in recumbency [mean 225.37 ± 60.78 pg/ml (624.29 ± 168.25 pmol/liter); N: 20–130 pg/ml (55–360 pmol/liter)] with suppressed renin concentration in the upright position [mean 2.87 ± 0.69 pg/ml (0.068 ± 0.0164 pmol/liter); N: 7–40 pg/ml (0.166–0.948 pmol/liter)]. In four patients, the diagnosis of adrenocortical adenoma was established by abdominal computed tomography scan and unresponsiveness of aldosterone to saline loading and confirmed by histological examination of the tissue after unilateral adrenalectomy, which led to normalization of blood pressure, plasma aldosterone, and potassium levels. In the other four patients, aldosterone was partially suppressed by sodium loading, and abdominal computed tomography scan did not reveal any adrenal tumor, supporting the diagnosis of IH. None of the subjects was taking any medication that could have influenced the renin-angiotensin-aldosterone system.

All the patients maintained a normal sodium diet (100–200 mmol per 24 h) throughout the study. SR 49059 [(2S) 1-[(2R 3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide] was supplied by Sanofi Research (Toulouse, France) as 100-mg tablets. SR 49059 is a potent and selective orally effective nonpeptidic antagonist of both rat and human V1a receptors. SR 49059 has an apparent half-life of 24–30 h in human and is rapidly and widely distributed in the body after a single oral dose administration (23). SR 49059 and placebo tablets were identical in appearance.

Study protocol

SR 49059 and placebo were administered orally to all patients (two tablets, once a day at 1100 h) in a double-blind fashion during two 1-wk treatment periods performed in a random order at 2-wk intervals. The tablets were administered at 1100 h to avoid fluctuations of ACTH secretion during the study, which could have influenced aldosterone secretion. For each treatment period, blood samples for measurements of aldosterone, renin, cortisol, ACTH, and electrolytes were obtained just before and 1, 1.5, and 2 h after SR 49059/placebo administration on d 1, 2, 3, and 7. All patients were maintained in a recumbent position from 0900 to 1300 h except on d 3 when the tests were performed in upright position.

For measurements of creatinine, electrolytes, and aldosterone concentrations, 24-h urines were collected on the day preceding each treatment period and on d 1, 2, 3, and 7 of the two treatments.

Hormone assays

Blood samples for ACTH were centrifuged at 4 C for 20 min. The plasma was separated and stored at –20 C until assay. ACTH levels were determined by immunoradiometric assay (Nichols Institute Diagnostic, Paris, France). Plasma aldosterone, cortisol, and renin levels were measured in duplicate using the following commercial kits: RIA (Beckman Coulter, Villepinte, France), Immunoluminescence (Diagnostic Product Corp., La Garennes-Colombes, France), and immunoradiometric assay (Bio-Rad, Marnes-La-Coquette, France), respectively. Intra- and interassay variations were lower than 9 and 11%, respectively, for all assays.

Statistical analysis

Statistical evaluation was performed by ANOVA. For patients with APA, aldosterone response to orthostatism was evaluated by calculating the amplitude of the peak of aldosterone expressed as absolute hormone level. The Fischer’s least significant difference multiple comparison test was used to analyze the differences between the SR49059- and placebo-treated patients. P < 0.05 was considered to be statistically significant. Correlation between plasma aldosterone and ACTH levels was calculated using the Pearson’s correlation test.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Immunohistochemistry

Immunoperoxidase labeling revealed the presence of AVP-stained cells in the APA tissues (Fig. 1AGo). AVP-like immunoreactivity was observed in two distinct types of cells (Fig. 1BGo). The majority of AVP-positive cells (80–85%), arranged as small clusters disseminated in the tumor tissue, exhibited the morphological characteristics of spongiocytic cells (24), i.e. large cells with abundant cytoplasm loaded with lipid inclusions (Fig. 1BGo, left). The staining was restricted to a limited area of the cytoplasm at the periphery of lipid droplets. A few smaller immunopositive cells (15–20%) scattered between the lobules of steroidogenic cells, had a chromaffin-like appearance with intense granular staining (Fig. 1BGo, right). Double-immunofluorescence labeling of APA slices with a monoclonal antibody against AVP and an antiserum against WE14 revealed that the small granular AVP-staining cells also contained chromogranin A, a marker of chromaffin cells (Fig. 1Go, D–F).


Figure 1
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  		FIG. 1. Immunohistochemical localization of AVP in APA. A, Low-magnification photomicrograph showing clusters of AVP-positive cells in APA. AVP-like immunoreactivity was revealed by the 3,3'-diaminobenzidine chromogen. A few examples of stained cells are shown (arrows). B, Higher-magnification photomicrographs showing AVP-positive material in the cytoplasm of spongiocytic (left) and chromaffin-like (right) AVP-positive cells. C, Incubation of tissue sections with AVP antibody preabsorbed with AVP (10–5 M) resulted in complete loss of the immunostaining. D–F, Dual-channel confocal laser scanning microscope analysis comparing the distribution of AVP and chromogranin A-like immunoreactivity in the APA. D, AVP-expressing cells were labeled with a monoclonal antibody against AVP and revealed with Alexa-488-conjugated goat antimouse {gamma}-globulins (GAM/Alexa 488). E, Chromogranin A-containing cells were labeled with a rabbit antiserum against WE14 (corresponding to the amino acid sequence 324–337 of chromogranin A) and revealed with Alexa-568-conjugated goat antirabbit {gamma}-globulins (GAR/Alexa 568). F, Overlay of the two images acquired in D and E, showing that three cells contain simultaneously AVP- and chromogranin A-like immunoreactivity. Scale bars, 50 µm (A–C), 20 µm (D–F).

 
Preincubation of the AVP antibody with synthetic AVP (10–5 M) totally suppressed the immunostaining (Fig. 1CGo). No labeling was observed when the AVP antibody was substituted with PBS (data not shown).

Pharmacological and molecular characterization of the AVP receptor expressed in APA

Effect of a V1a receptor antagonist on aldosterone secretion by perifused tumor tissue. The effect of AVP on aldosterone secretion by perifused adrenocortical tumor explants is shown in Fig. 2Go. Administration of AVP (10–7 M, 20 min) to the tissues induced a robust increase in aldosterone production (Fig. 2AGo). The maximum effect (+135%) on aldosterone output was observed 30 min after the beginning of AVP infusion. Prolonged administration of the specific V1a receptor antagonist [d(CH2)5,Tyr(OMe)2]AVP did not affect spontaneous aldosterone production but totally abrogated AVP-evoked aldosterone secretion (Fig. 2BGo).


Figure 2
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  		FIG. 2. Characterization of the AVP receptor expressed in APA. A, Effect of AVP (10–7 M, 20 min) on aldosterone secretion by perifused APA fragments. B, Effect of AVP during infusion of the V1a antagonist [d(CH2)5,Tyr(OMe)2]AVP (10–6 M) on aldosterone secretion. A single pulse of AVP (10–7 M, 20 min) was administered 40 min after the beginning of V1a antagonist infusion. The profile represents the mean secretion pattern of two independent perifusion experiments obtained from one Conn’s adenoma. Each point is the mean aldosterone production (expressed as a percentage of spontaneous steroid output) of two consecutive fractions collected during 5 min. The spontaneous level of aldosterone release (100% basal level) was calculated as the mean of eight consecutive fractions (40 min; {circ}) just preceding the administration of the secretagogue. The basal level of aldosterone in this experiment was 2.06 ng/g wet tissue per minute.

 
Expression of the V1a receptor in APA. The occurrence of V1a receptor mRNA was investigated after RT-PCR amplification in 18 APAs, using specific primers for the human V1a receptor. PCR products with the expected molecular size (505 bp) were detected in 11 of the 18 adenomas, whereas GAPDH mRNA was detected in all adenomas (data not shown). The cDNA bands were excised, ligated into p Gem-T, and sequenced. All sequences corresponded to the published sequence of the human V1a receptor cDNA.

In vivo study

In the eight patients with PA, oral administration of 200 mg SR 49059 had no influence on basal aldosterone (Fig. 3Go) or renin, ACTH, cortisol and K levels, or blood pressure (data not shown). As expected, no stimulation of aldosterone secretion to the upright posture was noticed in APA patients receiving placebo. However, in the same patients treated by SR 49059, the posture test induced a marked increase in plasma aldosterone levels (Fig. 4AGo). As shown in Fig. 4BGo, the peak amplitude measured during the orthostatism test in APA was significantly higher during SR 49059 treatment than those measured under placebo treatment (P < 0.03). Plasma renin levels remained suppressed throughout the duration of the test, and no modification of plasma ACTH and cortisol levels was observed. In patients with IH, SR 49059 had no effect on the aldosterone response to upright position.


Figure 3
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  		FIG. 3. Effect of the V1a antagonist SR 49059 and placebo on plasma aldosterone levels in the eight patients with PA. A single dose of 200 mg of SR 49059 ({blacksquare}) or placebo ({square}) was administered orally once daily during two 1-wk periods, in a random order, at 2-wk intervals. The effects of SR 49059 and placebo are shown in recumbent position on d 1, 2, and 7. Every day, blood samples were obtained at t0 (1100 h), t60, t90, and t120 min. Aldosterone is expressed as a percentage of basal level using a semilogarithmic scale.

 

Figure 4
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  		FIG. 4. Effect of the V1a antagonist SR 49059 ({blacksquare}) and placebo ({square}) on plasma aldosterone in the four patients with APA. A single dose of 200 mg SR 49059 or placebo was administered orally. A, The effects of SR 49059 and placebo are shown in both recumbent (d 1, 2, and 7) and upright (d 3) positions. See Fig. 3Go for other designations. B, Peak amplitude of the aldosterone response to the posture test performed on d 3 in patients taking SR 49059 or placebo is expressed as mean ± SEM, picograms per milliliter (picomoles per liter x 2.77). *, P < 0.03 vs. placebo.

 
No correlation was found between plasma aldosterone and ACTH in the IH group whatever the treatment (data not shown). In the four patients with APA, a significant correlation was observed between plasma aldosterone and ACTH levels (r = 0.33, P < 0.01; Fig. 5AGo), and this correlation was strengthened by the administration of SR 49059 (r = 0.67, P < 0.001; Fig. 5BGo).


Figure 5
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  		FIG. 5. Correlation between plasma aldosterone and ACTH levels in patients with APA receiving placebo (r = 0.33; P < 0.01) (A) and the V1a antagonist SR 49059 (r = 0.67; P < 0.001) (B).

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
In PA, the mechanisms involved in the maintenance of autonomous aldosterone secretion in the absence of circulating angiotensin II remain to be elucidated. We hypothesized that AVP, which is physiologically produced within the adrenal gland, may play a role in the pathogenesis of aldosterone hypersecretion.

Although the mRNA encoding preproAVP has been previously detected in APA (25), the presence of AVP in these tumors has never been reported. Here we show the occurrence of AVP-immunoreactive cells disseminated in the APA tissue. This observation indicates that, in aldosterone-producing tumors, AVP is stored not only in chromaffin cells, as in the normal adrenocortical tissue (8), but also in steroidogenic cells. The detection of AVP in spongiocytic cells provides further evidence for the presence of hybrid cells, i.e. exhibiting both steroidogenic and neuroendocrine characteristics in APA (26). In addition, the presence of AVP-secreting cells in APA tissue suggests that AVP, released by intratumoral cells, may exert an autocrine and/or paracrine stimulation of aldosterone secretion. We thus investigated the sensitivity of adenomatous cells to AVP by means of a perifusion system technique. In agreement with previous studies conducted on normal human adrenocortical tissue (9), we found that AVP is able to stimulate aldosterone production from perifused APA fragments. The stimulatory effect of the peptide was totally abolished by the selective V1a vasopressinergic receptor antagonist [d(CH2)5,Tyr(OMe)2]AVP, demonstrating the involvement of the V1a subtype receptor in the steroidogenic response to AVP, as previously observed in normal glomerulosa cells (9) or hyperplastic or tumor fasciculata tissues (15, 17, 27, 28). Consistent with earlier observations (25), we also found the presence of V1a PCR products in most APAs studied. Taken together, these results suggest that intratumoral AVP may stimulate aldosterone secretion through an autocrine/paracrine mechanism and that V1a vasopressinergic receptor antagonists may prove useful to reduce plasma aldosterone levels in patients with PA.

We then studied the effect of SR 49059, a nonpeptidic antagonist of the V1a receptor, on aldosterone secretion in eight patients with PA, i.e. four with APA and four with IH. SR 49059 did not induce any effect on basal plasma aldosterone levels in patients with APA or patients with IH, indicating that AVP does not play a key role in the maintenance of basal steroidogenesis in adrenocortical aldosterone-secreting lesions. Surprisingly, we observed that SR 49059 induced a sensitization of APA to posture. Indeed, plasma aldosterone level (peak height) rose in response to the upright stimulation test in patients with APA treated with SR 49059, whereas, as expected, it did not increase in the same subjects receiving placebo (29). Consistent with previous findings (30), we also found a positive correlation between plasma aldosterone and ACTH levels in patients with APA, a type of tumor that is known to overexpress ACTH receptor mRNA (31). Interestingly, this correlation was strengthened after SR 49059 administration. Collectively, these data indicate that SR 49059 modifies the tumor cell sensitivity to different stimuli including posture-sensitive hormones and ACTH. Among posture-sensitive hormones, catecholamines have been shown to stimulate mineralocorticoid production through activation of ß-adrenergic receptors that are, like the ACTH receptor, positively coupled to adenylyl cyclase (32, 33, 34). We suggest that, in adenomatous cells, AVP may diminish the aldosterone response to regulatory signals acting through the cAMP pathway. In agreement with this hypothesis, AVP has been shown to inhibit the aldosterone response to ACTH in rat glomerulosa cells (35). The effect of AVP on ACTH- and catecholamine-evoked aldosterone production likely involved a V1a receptor-mediated inhibition of calcium-sensitive adenylyl cyclase isoforms, which are expressed in the human zona glomerulosa (36). In the present study, the V1a receptor antagonist SR 49059 may therefore have restored or increased the sensitivity of aldosteronoma cells to catecholamines and ACTH respectively, through inhibition of the autocrine/paracrine action of AVP. In addition, the fact that SR 49059 modifies the secretory activity of APA indicate that AVP actually exerts a regulatory tone on aldosterone secretion in these tumors.

In conclusion, our results show the occurrence of AVP-containing cells in APA and the expression of functional eutopic V1a receptors in the tumor tissue. Our data also suggest that intraadrenal autocrine and/or paracrine factors and their receptors may represent new targets for medical therapies in Conn’s syndrome.


   Acknowledgments
 
The monoclonal antibody against AVP was a generous gift from Dr. Arlette Burlet (Equipe Associée 3453, Nancy, France), and the rabbit antiserum against WE14 was kindly provided by Drs. Youssef Anouar and Maité Montero-Hadjadje (Institut National de la Santé et de la Recherche Médicale Unité 413, Mont-Saint-Aignan, France). We thank Annick Legrand for biological measurements and Jean-François Menard for statistical analysis.


   Footnotes
 
This work was supported by Institut National de la Santé et de la Recherche Médicale Unité 413, Centre d’Investigation Clinique 204, Institut Fédératif de Recherche Multidisciplinaires sur les Peptides 23, the Centre Hospitalier Universitaire de Rouen, the Réseau COMETE (Cortico et Medullo: Etude des Tumeurs Endocrines, Programme Hospitalier de Recherche Clinique AOM 95201), the Plate-Forme Régionale de Recherche en Imagerie Cellulaire, and the Conseil Régional de Haute-Normandie. The in vivo study was supported by grants from Sanofi Research.

First Published Online January 31, 2006

Abbreviations: APA, Aldosterone-producing adenoma; AVP, vasopressin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IH, idiopathic hyperaldosteronism; PA, primary aldosteronism; RT, reverse transcription.

Received June 30, 2005.

Accepted January 23, 2006.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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