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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 6 2195-2203
Copyright © 1999 by The Endocrine Society

Original Studies

Vasopressin Receptors in Human Adrenal Medulla and Pheochromocytoma1

Eric Grazzini, Christophe Breton, Sylvain Derick, Miriam Andres, Danièle Raufaste, Frédérique Rickwaert, Gilles Boccara, Pascal Colson, Nathalie C. Guérineau, Claudine Serradeil-Le Gal and Gilles Guillon

INSERM U-469, CCIPE (E.G., C.B., S.D., N.C.G., G.G.), 34094 Montpellier Cedex 05; SANOFI Recherche Toulouse (D.R., C.S.-L.G.), 31036 Toulouse; and Département d’Anesthésie, Réanimation B, Hôpital Arnaud de Villeneuve (F.R., G.B., P.C.), 34295 Montpellier, France

Address all correspondence and request for reprints to: Gilles Guillon, INSERM U-469, Centre CNRS-INSERM de Pharmacologie Endocrinologie, 141 rue de la Cardonille, 34094 Montpellier Cedex 05, France. E-mail: guillon{at}u469.montp.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The nature of vasopressin (VP) receptors present in normal and tumoral human adrenal was investigated using various experimental approaches. Specific VP-binding sites were detected by autoradiography using [3H]arginine VP as a radioligand in adrenal cortex and medulla. The V1a receptor subtype was expressed in the two parts of the gland, as shown by pharmacological studies and RT-PCR experiments. By contrast, the V1b receptor subtype was only expressed in medullary chromaffin cells. This was confirmed by the characterization of V1b transcripts detected in adrenal medulla tissues. In pheochromocytoma, we also detected functional V1b receptors. These receptors triggered intracellular calcium mobilization from intracellular pools and were involved in catecholamine secretion. Binding experiments performed on pheochromocytoma plasma membrane preparations also revealed V1a vasopressin-binding sites, whose roles and cellular localization have not yet been determined. RT-PCR experiments confirmed these data; 100% and 80% of the five tumors tested exhibited V1a and V1b transcripts, respectively. Perifusion experiments also demonstrated that some pheochromocytomas may secrete large amounts of VP. Our findings imply that VP locally secreted by human adrenal medulla may regulate adrenal function by acting on V1a or V1b receptors. More interestingly, we demonstrate that one pheochromocytoma oversecretes VP. In this particular case, this may contribute to the increase in blood pressure observed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MAMMALS, vasopressin (VP) is synthesized by specific neurons of the supraoptic or paraventricular nucleus and is released in the portal blood circulation at the pituitary level (1). VP exerts three main physiological functions: contraction of vascular smooth muscles, antidiuresis in the kidney, and secretion of ACTH in the anterior pituitary. These different effects are triggered by three distinct VP receptor subtypes, V2, V1a, and V1b, coupled to at least three distinct transduction mechanisms: the adenylyl cyclase pathway for the V2 receptor, the inositol trisphosphate and diacylglycerol pathway for the V1a and the V1b receptors, and the activation of calcium influx for the V1a receptor (for review, see Refs. 2, 3).

VP receptors have been characterized in many peripheral organs and in brain (2, 3). Given the affinity of these receptors for VP (0.5–5 nmol/L), the low concentration of plasma VP (0.1–5 pmol/L) raises questions about their physiological roles (for review, see Ref. 4). The coexpression of VP receptors and its ligand in many tissues, such as ovaries, testis, pancreas, and vascular smooth muscle cells, supports the hypothesis that this peptide may act through an autocrine/paracrine mechanism (5, 6, 7). This was further confirmed in the rat adrenal gland, where VP is synthesized and secreted by the medulla. This secretion is stimulated by neurotransmitters (acetylcholine) or neuropeptides such as corticotropin-releasing factor (CRF), which is known to be present in the medulla (4). Functional VP receptors have been also characterized in the adrenal gland. In the adrenal cortex, by acting on the V1a VP receptor subtype, VP exerts two main effects: it increases the mitogenic activity of the zona glomerulosa in the rat (8) and stimulates aldosterone and cortisol secretions in rat, cat, and calf zona glomerulosa or in calf zona fasciculata, respectively (see Refs. 4, 9 for review; 10, 11). In the medulla, Antoni and Taylor first demonstrated the presence of specific VP receptors. Their pharmacological profile and physiological function are not clearly identified (12, 13). More recently, we demonstrated that V1b VP receptors are expressed in rat adrenal medulla. These receptors, localized in chromaffin cells, stimulate catecholamine secretion and may also be involved in ACTH secretion (14, 15).

The regulation of human adrenal medulla by VP has been poorly studied. We have recently shown that VP is released by human medulla (16). In human adrenal cortex, only V1a receptors involved in aldosterone and cortisol secretion have been characterized (16, 17). Physiopathological studies also revealed that Cushing’s syndrome caused by ACTH-independent tumors or nodular adrenal hyperplasia can be accounted for by an overresponsiveness to VP responsible for hypercortisolism (18). Perraudin and collaborators (19) and Lacroix and collaborators (20, 21) also suggested that such tumors might overexpress an eutopic V1a VP receptor or express a mutated form of the V1a receptor exhibiting an oversensitivity for VP. Several studies also indicated that human pheochromocytoma might overexpress VP, as immunoreactive VP has been found in various adrenal medulla tumors (22, 23), and high concentrations of plasma VP have been measured during pheochromocytoma excision (24).

The aim of this study is to characterize the VP receptor subtypes expressed in human adrenal medulla and pheochromocytoma. We addressed the question of a possible overexpression of VP or eutopic VP receptors in medulla tumors, as previously observed in some neuroendocrine tumors (25).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals

The chemicals used in the present study were obtained from the following sources. [3H]arginine VP ([3H]AVP; 60 Ci/mmol) was obtained from New England Nuclear (Les Ulis, France). [Arg8]VP antiserum and [125I]Arg8-VP were purchased from Amersham (Les Ulis, France). d[D-3-Pal]VP, a V1b agonist (26), and OH-LVA, a linear vasopressin antagonist exhibiting a very good affinity for V1a receptors, were gifts from Dr. M. Manning (Toledo, OH). This analogue was radioiodinated by the iodogen technique as previously described with a specific radioactivity of 2000 Ci/mmol (27). SR 49059, a specific nonpeptidic antagonist of V1a VP receptor was synthesized by SANOFI Laboratory (28). Collagenase, HBSS, Eagle’s MEM, Trizol, Moloney murine leukemia virus reverse transcriptase, and Taq polymerase were purchased from Life Technologies (Eragny, France). Deoxyribonuclease and fura-2/AM were obtained from Sigma Chemical Co. (St. Quentin Fallavier, France). AVP and acetylcholine were obtained from Bachem (Voisins le Bretonneux, France), and the dideoxy chain termination kit was purchased from Pharmacia Biotech (France). Fluo 3/AM and pluronic F127 were from Molecular Probes (Eugene, OR). All other reagents were of A-grade purity. Dopamine ß-hydroxylase antibody was obtained from Chemicon (Euromedex, France).

Origin of human adrenal glands and human pheochromocytoma

Adrenal glands were obtained at the time of either renal transplantation or nephrectomy from renal carcinoma from patients, aged 20–60 yr, and pheochromocytoma at the time of adrenalectomy. Immediately after excision, glands without microscopic abnormalities or tumors were stored at 4 C and dissected within a delay of 1–6 h. Glands were either frozen at -40 C in isopentane, as previously described (16), and further stored at -80 C for autoradiographic studies or dissected to prepare crude plasma membranes for binding studies (16). This project was approved by the human subject review committees of our institutions and was carried out in collaboration with Hôpital Lapeyronie (Montpellier, France).

Autoradiography

Slices (15 µm) from frozen human adrenal were mounted on gelatin chrome alum slides, rinsed to eliminate endogenous AVP, and incubated with 1 nmol/L [3H]AVP with (nonspecific binding) or without (total binding) 1 µmol/L unlabeled AVP, as previously described (16). Slices were then rinsed twice for 5 min each time in an ice-cold solution composed of 50 mmol/L Tris-HCl, pH 7.4, plus 3 mmol/L MgCl2 and dipped briefly in ice-cold distilled water. After drying under a stream of cold air, slices were placed on a phosphorimaging plate specific for tritium (Fuji Photo Film Co., Ltd., Tokyo, Japan) for 4–10 days. The exposed imaging plates were scanned and digitalized to quantify binding receptor density. Dose-displacement curves of specific binding were calculated from these digitalized images, as a function of the amount of the unlabeled VP analogues added together with [3H] AVP in the incubation medium. The IC50 value, defined as the concentration of analogue required to obtain 50% inhibition of the specific binding, was calculated, and the inhibition constant (Ki) values were determined from IC50 values using the Cheng and Prusoff equation. Data for competition experiments were analyzed using an iterative nonlinear regression program (29).

Binding assays on plasma membrane preparations

Specific binding of either [3H]AVP or [125I]OH-LVA was performed, as previously described, on crude plasma membrane preparations derived from at least three distinct human adrenal glands (zona glomerulosa) or human pheochromocytoma (16). The amount of labeled hormone that bound to membranes was determined by filtration through Whatman GF/C filters (Clifton, NJ). All determinations were performed in triplicate, and nonspecific binding was determined in the presence of 1 µmol/L unlabeled AVP.

Cell primary culture

The adrenal medulla was dissected under binocular loop and separated from the zonae fasciculata/reticularis, taking advantage of the distinct color of each region (brown for the cortex, white for the medulla). Adrenal medulla was then digested with collagenase and mechanically dispersed, as previously described for rat adrenal medulla (14). The cells obtained were plated on a polyornithine-coated glass coverslip and grown for 1 day at 37 C in a humidified atmosphere (95% air-5% CO2) Medulla cell primary culture (MC) purity was determined using two criteria. Firstly, microscope examination revealed principally the presence of small round refringent cells, very different from glomerulosa or fasciculata cells, which exhibited a polygonal morphology and an abundant content of refringent lipid droplets. Moreover, MC did not divide, in contrast to zona glomerulosa cells primary culture (ZGC). Secondly, we characterized MC by measuring their abilities to respond to acetylcholine, because chromaffin cells exhibited functional nicotinic receptors at variance with glomerulosa cells. Using the calcium video technique, we demonstrated that acetylcholine never altered the ZGC intracellular Ca2+ concentration ([Ca2+]i). In contrast, 57 ± 4% of MC responded to this agonist (n = 50 cells originating from three distinct primary cultures; data not shown).

Human pheochromocytoma cell primary culture (PC) was performed as described for human medulla. Cell culture purity was determined as it was for MC by measuring their abilities to respond to nicotinic receptor agonist. Using the calcium videomicroscopy technique, we found that 87% of cells exhibited a positive response to 100 µmol/L nicotine (n = 55 cells originating from two distinct primary cultures).

Measurement of [Ca2+]i in individual cells

MC were loaded with 3.3 µmol/L fura-2/AM (45 min, 37 C) in serum-free culture medium supplemented with 20 mmol/L HEPES, pH 7.4. Cells were then washed three times in HBSS medium and stimulated at room temperature with various secretagogues. Details of the experimental procedures allowing the determination of [Ca2+]i had been previously described (14). In some experiments, to be sure that only chromaffin cells were studied, only cells positively responding to acetylcholine or nicotine were selected.

PC were loaded with 10 µmol/L fluo-3/AM and 10 µmol/L F127 (45 min, 37 C) in serum-free culture medium as previously described (30). Fluo-3 was excited through a 488-nm band-pass filter, and the emitted fluorescence was collected through a 515-nm barrier filter. Because fluo-3 is a single wavelength dye, its emission is a function of both intracellular Ca2+ and dye concentration. [Ca2+]i changes were therefore expressed as the F/Fmin ratio, where Fmin is the minimum fluorescence intensity measured during the recording.

RT-PCR analysis

Total ribonucleic acid (RNA) from adrenal tissues was extracted using Trizol (Life Technologies); 3 µg RNA were reverse transcribed into complementary DNA (cDNA) using random hexamers and Moloney murine leukemia virus RT (Life Technologies) as previously described (31). For the V1b amplification, the primer pair (27-mer) was designed to amplify a 254-bp cDNA fragment (residues 265–349) (32). For the V1a amplification, the primer pair (26-mer) generated a 468-bp cDNA fragment (residues 253–408) (33). As an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA also was amplified using a primer pair (26-mer) design to amplify a 470-bp region of GAPDH (34). The PCR products were subcloned using pGEM-T easy vector systems (Promega Corp., Charbonnieres, France) and sequenced to verify the specificity of the amplification sequences.

Perifusion experiments

Fragments (50 mg) of freshly dissected pheochromocytoma were mixed with 0.4 g Bio-Gel, Bio|b1Rad, Ivry sur Seine, France perifused at 37 C as previously described (16) with or without the secretagogues to be tested. VP in the perifusion fractions was measured by RIA using [Arg8]VP antiserum as previously described (14, 16).

Exocytotic site detection

To assess catecholamine secretion from PC, we used an indirect immunofluorescent method, as previously described (14). This approach consisted of measuring dopamine-ß-hydroxylase (DßH) translocation from intracellular secretory vesicles to plasma membrane. Briefly, cells were fixed with 3.7% formaldehyde, extensively washed, and incubated for 1 h at room temperature with an antibody directed against rabbit DßH (1:200 dilution). Cells were further rinsed and incubated for 1 h at room temperature with a goat antirabbit fluorescein isothiocyanate-conjugated (1:60 dilution). Fluorescence was observed using a Zeiss epifluorescence microscope (Carl Zeiss, New York, NY).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Localization and pharmacological characterization of VP-binding sites in human adrenal glands

Autoradiography experiments using [3H]AVP as radioligand were performed to detect all of the VP receptor subtypes expressed in human adrenal glands. As illustrated in Fig. 1Go, specific binding sites were detected not only in adrenal cortex (zona glomerulosa and zona fasciculata), but also in the medulla. By this technique, it was not possible to determine the density of VP receptors in terms of picomoles of [3H]AVP bound specifically per mg tissue. However, we could compare the relative labeling of the different parts of the gland, as the affinities of [3H]AVP for the different VP receptors are similar (16), and the autoradiographic technique used has a wide linear dynamic range (29). Calculations performed on three distinct glands indicated that the specific labeling of the medulla obtained with 1 nmol/L [3H]AVP represents 38 ± 7% of that measured on the cortex of the corresponding slice.



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  		Figure 1. Localization of [3H]AVP-binding sites in human adrenal gland by autoradiography. Slices of human adrenal gland were incubated for 1 h at room temperature with 1 nmol/L [3H]AVP (total control binding), with the same amount of labeled VP and increasing concentrations of unlabeled SR 49059, or with 1 nmol/L [3H]AVP and 1 µmol/L unlabeled VP (nonspecific binding). After extensive washings, radioactivity associated with each slice was measured, and quantification of the radioactivity was performed as described in Materials and Methods. Gradation color bar shows lower and higher intensities of labeling.

 
Figures 1Go and 2Go illustrate dose-displacement experiments using a series of unlabeled VP analogues exhibiting a good selectivity for the different human VP receptor subtypes. SR 49059, a specific nonpeptidic V1a receptor antagonist (28), fully displaced specific [3H]AVP binding in both the adrenal cortex and the medulla (Fig. 1Go). IC50 and the subsequent dissociation constant (Ki) for VP receptor binding sites were determined by measuring the relative specific binding of small patches of cortex or medulla (Fig. 2Go). All unlabeled ligands tested dose dependently displaced all specific [3H]AVP binding. For data deriving from the adrenal cortex, the dose-displacement binding curves could be fitted with a single [3H]AVP-binding site model with a Hill number close to 1. The Ki values deduced from these calculations are summarized in Table 1Go. They confirm the presence of a V1a receptor subtype in the adrenal cortex, as SR 49059 totally displaced all the specific binding with a Ki of about 1 nmol/L, and d[D-3-Pal]VP and dDAVP were much less potent. Moreover, these values were in good agreement with those previously detected on membrane preparations derived from human adrenal cortex (16). Such data validated the autoradiographic technique used to determine the Ki of unlabeled analogues. The dose-displacement curves deriving from adrenal medulla could also be fitted using a single [3H]AVP-binding site model for dDAVP and d[D-3-Pal]VP. Using SR 49059, we obtained a biphasic displacement on one gland of three tested (see profile of SR 49059 relative to adrenal medulla; Fig. 2Go). For the other two glands studied, the dose-displacement curves obtained were not different from those obtained with SR 49059 on adrenal cortex (data not shown). The Ki obtained for the different VP analogues tested are summarized in Table 1Go. They were similar to those calculated for adrenal cortex, suggesting that adrenal cortex and adrenal medulla both express the V1a VP receptor subtype.



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  		Figure 2. Characterization of VP receptors present in human adrenal gland. Autoradiography experiments were performed, as illustrated in Fig. 1Go, by incubating human adrenal slices with labeled [3H]AVP and increasing amounts of unlabeled VP analogues ({circ} and •, SR 49059; {triangleup} and {blacktriangleup}, dDAVP; {square} and {blacksquare}, d[D-3-Pal]VP). The relative intensity of specific binding was determined either on small patches of adrenal cortex (•, {blacktriangleup}, and {blacksquare}) or on adrenal medulla ({circ}, {triangleup}, and {square}) by computer analysis (specific binding = total binding measured in the presence of a given concentration of an unlabeled VP analogue - nonspecific binding measured in a similar area of an adjacent slice). These relative intensities were expressed as a percentage of control specific binding (specific binding measured in a similar area of an adjacent slice in the absence of unlabeled VP analogueues).

 

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  		Table 1. Affinities of vasopressin analogues for human adrenal vasopressin receptors

 
Pharmacological characterization of VP-binding sites in human pheochromocytoma

Crude plasma membranes derived from human pheochromocytoma also expressed specific VP-binding sites. Figure 3AGo illustrates a typical Scatchard representation of a dose-dependent binding curve using [125I]OH-LVA. A single class of binding site was detected. Statistical values derived from three distinct pheochromocytoma membrane preparations indicated that the specific binding sites detected exhibited an affinity similar to that described for the human V1a receptor (Kd = 20.3 ± 3.3 pmol/L) (16). The maximal binding capacity detected was low (Bmax = 11.7 ± 4.5 fmol [125I]OH-LVA specifically bound/mg protein). Competition experiments using specific unlabeled vasopressin analogues confirmed a V1a pharmacological profile: SR 49059 and CVPA displaced [125I]OH-LVA with a good affinity (Ki = 0.9 ± 0.4 and 8.0 ± 2.7 nmol/L, respectively; three distinct determinations). By contrast, OT and dDAVP were less efficient (Ki = 356 and 300 nmol/L, respectively; two distinct determinations). Using [3H]AVP, specific binding sites were also characterized on the same pheochromocytoma membrane preparations (Kd = 1.7 ± 0.6 nmol/L; Bmax = 27 ± 18 fmol [3H]AVP bound/mg protein; three distinct determinations). Their high affinity for SR 49059 confirmed that [3H]AVP binds to the V1a receptor (Ki = 0.4 ± 0.1 nmol/L; four distinct determinations; data not shown).



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  		Figure 3. Characterization of VP receptors present in human pheochromocytoma membrane preparations. A, Saturation experiments. Crude plasma membranes derived from human pheochromocytoma (50–100 µg protein/assay) were incubated for 45 min at 37 C in the presence of increasing amounts of [125I]OH-LVA (Free) with (nonspecific binding) or without (total binding) 0.2 µmol/L unlabeled VP as described in Materials and Methods. Specific binding (Bound) was calculated in each condition and plotted against the bound/free ratio. B, Competition experiments. Pheochromocytoma plasma membranes were incubated as described in A but in the presence of a constant amount of [125I]OH-LVA (20 pmol/L) with or without (control) increasing amounts of unlabeled VP analogueues. Specific binding was calculated in each condition and expressed as a percentage of the control values (100% = 450 ± 50 cpm/assay).

 
Functional characterization of VP receptors in normal (MC) and tumoral (PC) human adrenal medulla cell primary cultures

The presence of functional VP receptors on MC were investigated using the calcium videomicroscopy tech-nique. Figure 4AGo illustrates VP and acetylcholine-induced [Ca2+]i changes. Both agonists induced a rapid increase in [Ca2+]i within a few seconds (transient phase). Thereafter, [Ca2+]i weakly decreased and remained stable for a few minutes (plateau phase). By sequentially applying the two effectors on the same cell, it was possible to determine the percentage of MC sensitive to a given stimulus. Data obtained from at least three distinct MC primary cultures indicated that 60% and 57% of cells responded to VP and acetylcholine, respectively, and 40% responded to both stimuli. To further study the [Ca2+]i change induced by VP in MC, we tested the role of external calcium. As shown in Fig. 4BGo, decreasing the free external calcium concentration from 1.8 mmol/L to 0.1 µmol/L reduced, but did not fully suppress, the transient phase. Similarly, the plateau phase was reduced, and [Ca2+]i returned to basal values within 2–3 min. As shown in Fig. 4CGo, VP increased [Ca2+]i in a dose-dependent and saturable fashion. AVP (1 nmol/L) began to stimulate [Ca2+]i, and maximal effects were observed for 30 nmol/L. The concentration of AVP leading to a half-maximal effect (ED50) was 3.0 ± 2.0 nmol/L (three distinct experiments). To determine the nature of the VP receptor present on MC, we tested the influence of SR 49059 on the [Ca2+]i response induced by VP on both ZGC and MC. As illustrated in Fig. 5Go, whatever the cells considered, SR 49059 inhibited the hormonal response. Two types of effects could be distinguished. SR 49059 was 22-fold less active on MC sensitive to acetylcholine compared to unsensitive MC (Ki for SR 49059 = 45 ± 6 and 2.0 ± 0.5 nmol/L, respectively). In ZGC, SR 49059 inhibited the calcium response induced by VP with a Ki similar to those calculated for MC unsensitive to acetylcholine.



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  		Figure 4. Effect of VP on [Ca2+]i of human medulla cell primary culture. MC primary cultures grown 2 days on glass coverslips were loaded with fura-2/AM as described in Materials and Methods. A, Cells were incubated in HBSS medium containing 1.8 mmol/L free CaCl2, and [Ca2+]i was measured every 15 s before and after the addition of 100 nmol/L VP for 250 s ({circ}). Thereafter, the incubation medium was aspirated and replaced by fresh HBBS medium deprived of VP but containing 10 µmol/L acetylcholine. [Ca2+]i was then measured for an additional 250-s period ({blacktriangleup}). In B, similar experiments were performed, but cells were incubated in an HBSS medium containing either 1.8 mmol/L (•) or 0.1 µmol/L ({circ}) free CaCl2. Stimulation was induced by adding 0.1 µmol/L VP. C, MC primary cultures were stimulated with increasing amounts of VP, and [Ca2+]i was measured as described in A. The increase in [Ca2+]i was determined by integrating the kinetic curve of [Ca2+]i from 0–4 min and was plotted as a function of the concentration of hormone used. Results are the mean of [Ca2+]i originating from 15–30 individual VP- and acetylcholine-sensitive cells from one experiment, representative of three.

 


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  		Figure 5. Effect of SR 49059 on the VP-stimulated calcium response of human medullary cell primary cultures. MC ({triangleup} and {blacktriangleup}) and ZGC primary culture ({circ}) were loaded with fura-2/AM and preincubated for 15 min in an HBSS medium containing 1.8 mmol/L free CaCl2 with or without (control) increasing amounts of SR 49059. Cells were then stimulated with 30 nmol/L VP, and [Ca2+]i was measured every 15 s for 4 min. The increase in [Ca2+]i was determined as described in Fig. 4AGo. Cells were further stimulated with maximal dose of acetylcholine to determine the cells that were sensitive ({blacktriangleup}) and unsensitive ({circ} and {triangleup}) to this neurotransmitter. Results, expressed as the percentage of calcium mobilized from control cells, are plotted as a function of the concentration of SR 49059 used during the preincubation period. Data are the mean of 10–20 individual VP-sensitive cells originating from a single experiment, representative of three.

 
[Ca2+]i measurements performed on single pheochromocytoma cells in primary cultures also revealed the presence of functional VP receptors. Twenty-four percent of cells sensitive to nicotine (n = 55 from 2 distinct primary cultures) also responded to AVP (data not shown). As illustrated in Fig. 6AGo, d[D-3Pal]VP, a specific V1b agonist, was also able to increase [Ca2+]i in PC. To further verify the functionality of these receptors, we tested the abilities of different secretagogues to stimulate catecholamine secretion. In basal conditions, DßH was not associated to cell plasma membranes. By contrast, upon addition of 100 µmol/L nicotine, intense labeling was observed all around the PC, suggesting that the secretory vesicles that contain DßH fused within the plasma membrane (Fig. 6BGo). VP and d[D-3-Pal]VP produced the same effect. Upon VP stimulation, 26% of the cells exhibited a positive labeling to DßH (n = 231 cells from 2 distinct primary cultures originating from 2 distinct tumors).



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  		Figure 6. Functional characterization of VP receptors present on human pheochromocytoma cell primary cultures. Tumoral cells were grown for 1 day on glass coverslips as described in Materials and Methods. A, d[D-3-Pal]VP (1 µmol/L, 10 s)-induced [Ca2+]i changes recorded in a chromaffin cell loaded with fluo-3. In the same cell, further nicotine (100 µmol/L, 5 s) application triggered a typical increase in [Ca2+]i. All drugs were bath applied. B, Immunofluorescent detection of DßH in individual cells. Cells were incubated in the absence (control) or presence of d[D-3-Pal]VP (1 µmol/L) or nicotine (100 µmol/L) for 4 min. Exocytotic sites were only detected in stimulated cells, appearing as a fluorescent ring at the plasma membrane level.

 
Expression of VP receptor genes in human adrenal medulla and pheochromocytoma

To determine which VP receptor genes were expressed in normal and tumoral human adrenal medulla, we performed RT-PCR analysis of RNA extracted from five glands and four pheochromocytoma. The presence of GAPDH transcripts was also assessed as a control. As shown in Fig. 7Go, single bands of 254 bp (V1b), 468 bp (V1a), and 470 bp (GAPDH) were generated with specific primers, respectively. The sizes of the PCR amplification products corresponded to the sizes predicted from the genomic sequences. Using the specific V1b primers, the same band was obtained using total RNA from V1b-expressing CHO cells (lane 2), human adrenal medulla (lanes 3 and 5), and pheochromocytoma (lane 7). However, V1b amplification signal was observed in four adrenal medulla of five and in three pheochromocytoma of four, whereas V1a signal was detectable in all samples. The natures of the PCR products were further assessed after subcloning and sequencing the specific bands. cDNA sequences obtained for CHO stably transfected with the V1b receptor, adrenal medulla, and pheochromocytoma were the same and were identified as the sequence of human V1b vasopressin receptor. Similar results were obtained for V1a PCR products (data not shown).



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  		Figure 7. V1b receptor gene expression in human adrenal medulla and pheochromocytoma. RT-PCR was performed using no RNA (negative control; lane 1), 1 ng human V1b or V1a receptor expressing CHO cells RNA (positive control; lane 2), 3 µg adrenal medulla RNA (lanes 3–5), or 3 µg of pheochromocytoma RNA (lanes 6 and 7). PCR amplification was performed using either V1b or V1a primer pairs. As a cDNA control, PCR amplification was also performed using a primer pair specific for human GAPDH. The PCR products were separated by agarose gel electrophoresis, revealed by ethidium bromide, and photographed under UV light. DNA markers (1-kb ladder) were run in parallel. The sizes of the amplified products are indicated in base pairs.

 
Secretion of AVP by human pheochromocytoma

A young patient exhibiting sustained hypertension (220/110 mm Hg), weak urinary output (800 mL/day), hyponatremia (128 mmol/L) without dyskalemia, and left latero aortic pheochromocytoma tumor was scheduled to undergo tumorectomy. During surgery, adrenaline, noradrenaline, and dopamine concentrations did not vary markedly and remained in the normal range (35). By contrast, the plasma VP concentration measured during surgical manipulation just before tumorectomy was very high (220 pmol/L; normal range, 0.4–4 pmol/L). Thirty minutes after tumor excision, the plasma VP concentration dropped to 26 pmol/L, and arterial blood pressure returned to normal values. To test for possible hypersecretion of VP by the adrenal tumor, we performed perifusion experiments on one fragment of this pheochromocytoma. As illustrated in Fig. 8Go, the tumor spontaneously secreted VP at a mean rate of 1.8 fmol VP/mL perifusate. Upon stimulation by 100 nmol/L CRF, a secretagogue known to be synthesized in human adrenal medulla and to stimulate VP secretion (16, 23), the rate of VP secretion increased 8000-fold and returned to the baseline within 20 min.



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  		Figure 8. VP release by human pheochromocytoma. A freshly isolated pheochromocytoma was dissected, and 50 mg tissue were mixed with Bio-Gel P2 and superfused during a 140-min period without stimulation ({circ}) or with a 20-min stimulation with 100 nmoL/L CRF (•). Perifusate fractions were collected every 2 min, and VP content was determined by specific RIA as previously described (16 ).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In human adrenal glands, few data concerning the secretion of VP and the expression of its receptors are available. In this study, we characterized the VP receptors expressed in human adrenal medulla and compared these data to those found on human adrenal medulla tumors.

In human adrenal slices, binding studies performed using [3H]AVP, a tritiated ligand able to recognize all VP receptor subtypes (2, 3), revealed the presence of specific VP-binding sites in the medulla. These receptors exhibited a V1a pharmacological profile similar to that previously determined in the human cortex (16). For instance, specific peptidic (CVPA) and nonpeptidic V1a antagonists (SR 49059) (28) exhibited a high affinity for medulla VP-binding sites. RT-PCR experiments confirmed these results, as the mRNA encoding the human V1a receptor was detected in all human medullas tested. Calcium videomicroscopy experiments performed on acetylcholine-unsensitive MC confirmed the presence of functional V1a receptors; SR 49059 antagonized the calcium response induced by VP with a Ki around 1 nmol/L, as previously observed on ZGC (16).

The presence of V2 VP receptors in human adrenal medulla can be ruled out, because the affinity of dDAVP, a V2 agonist exhibiting a nanomolar affinity for the human V2 receptor and a Kd at least 10-fold higher for the V1a or V1b receptor subtype (36, 37), was around 64 nmol/L for human adrenal medulla VP-binding sites.

An interesting result obtained in one adrenal showed a biphasic dose displacement of [3H]AVP by SR 49059, suggesting the presence of more than one VP receptor subtype. As the V2 receptor subtype was not expressed in human adrenal medulla, we suspected the presence of V1b receptor. Calcium videomicroscopy experiments confirmed this hypothesis; SR 49059 antagonized the VP response of acetylcholine-sensitive MC with an efficiency similar to that measured on rat chromaffin cells known to exhibit only V1b receptor (14) and 30-fold lower than that observed on human ZGC known to express only the V1a receptor subtype (16). RT-PCR analysis indicated the presence of mRNA encoding for the V1b receptor in 80% of the human adrenal medullas tested.

Altogether, these data show that human adrenal medulla principally expresses the V1a VP receptor subtype and, to a lesser extent, the V1b subtype. As previously observed on rat adrenal gland, V1b receptors are exclusively present on chromaffin cells (MC sensitive to acetylcholine) (14). The cells of the adrenal medulla exhibiting the V1a VP receptors, however, remain to be identified. They correspond to MC that do not respond to nicotinic agonist. They probably derive from adrenal medulla arteries, as 1) endothelial cells and aortic myocytes express V1a VP receptors (38, 39); and 2) arteries from tissues not known to express V1a VP receptors are specifically and intensively labeled using [125I]OH-LVA (40).

Our results are in good agreement with previous studies performed on rat adrenal glands. V1a receptors are located on rat adrenocortical cells and in adrenal medulla tissue, whereas V1b are present only on chromaffin cells (9, 14). V1a receptors are also expressed in bovine adrenal medulla tissues, as specific V1a VP antagonist inhibited [3H]AVP binding with a good efficiency (12, 13). However, as found for SR 49059 in one human adrenal gland, the dose-displacement curve obtained with a specific V1a antagonist was curvilinear, suggesting that more than one VP receptor subtype is present in bovine adrenal medulla.

These data also indicate the role of VP receptors in mammalian adrenal medulla. In bovine chromaffin cells, stimulation with 1 µmol/L VP partially inhibits the nicotinic-stimulated catecholamine secretion, but does not modify the basal secretion (13). By contrast, in rat and human tumoral chromaffin cells, VP stimulates catecholamine secretion (Ref. 14 and this study). Such discrepancies may arise from the different techniques used for measuring catecholamine secretion (immunocytochemistry using DßH labeling vs. perifusion and RIA measurement) or from a difference between human and bovine species. Adrenal medulla VP receptors could also be involved in ACTH and CRF secretion, as demonstrated in the rat by Mazzochi and collaborators (15). This remains to be verified on normal and tumoral human adrenal medulla.

VP-binding sites from human pheochromocytoma membrane preparations were also characterized. Whatever the labeled ligand used, ([3H]AVP or [125I]OH-LVA), SR 49059 displaced the specific binding with a good efficiency (Ki = <=1 nmol/L), indicating the presence of the V1a VP receptor subtype. Such results were confirmed by RT-PCR experiments. The density of AVP receptors detected in human pheochromocytoma (25 fmol/mg protein) was similar to that found on normal human adrenal medulla, as we previously showed that plasma membrane preparations deriving from human adrenal cortex exhibited a density of 65 fmol [3H]AVP specifically bound/mg protein (16) and that the density of adrenal medulla VP-binding sites represented around 38% that in the adrenal cortex (this study). As determined for human adrenal medulla, binding experiments performed with [3H]AVP also failed to detect V1b-binding sites. However, both RT-PCR experiments and calcium videomicroscopy measurements revealed the presence of V1b receptors on PC. These cells are probably chromaffin cells, because they responded to nicotinic agonists and were labeled with DßH antibody, an enzyme involved in catecholamine synthesis.

In contrast with experiments performed on human bronchial carcinoids (25), our data do not reveal any overexpression of V1a or V1b receptors in human pheochromocytoma. However, by using PCR and binding approaches, we cannot rule out that eutopic V1a receptors exhibiting an overresponsiveness for VP may be present, as described in some ACTH-independent macronodular adrenal hyperplasia (19, 20, 21). More interestingly, our results indicate that some adrenal tumors may secrete large amount of VP. To our knowledge, this is the first clear characterization of VP-secreting pheochromocytoma. To date, previous reports only suggest this possibility, as immunoreactive VP has been found in many pheochromocytoma (22), and the plasma VP level of patients undergoing pheochromocytoma resection has been often found to be elevated (24).

In conclusion, we demonstrate that human adrenal medulla express both V1a and V1b receptors. More interestingly, we show that human adrenal medulla tumors do not overexpress these receptors, but may oversecrete VP, which may contribute to increasing blood pressure.


   Acknowledgments
 
We thank M. Chalier for typing and correcting the manuscript, M. Passama for preparing the illustrations, and J. P. Patacchini for helpful comments on the manuscript. We are also thankful to Maurice Manning for the gift of peptides, and to Nicole Gallo-Payet for fruitful discussion.


   Footnotes
 
1 This work was supported by grants from INSERM. Back

Received June 22, 1998.

Revised December 2, 1998.

Revised February 23, 1999.

Accepted March 2, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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