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Original Studies |
Groupe d’Etudes en Physiopathologie Endocrinienne, INSERM CJF 9208, Institut Cochin de Génétique Moléculaire; Laboratoire d’Explorations Fonctionnelles Endocriniennes, Hôpital Trousseau; and INSERM U-36, Collège de France; Paris; and Endocrinologie et Maladies Métaboliques, CHU de Rouen, Rouen, France
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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We conclude that the vasopressin V1 receptor gene is expressed in normal and tumoral adrenocortical cells. High, and not ectopic, expression occurs in a minority of tumors that become directly responsive to vasopressin stimulation tests.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Adrenocortical tumors have provided yet another pathogenetic concept: that of illegitimate receptor expression with the demonstration that gastric inhibitory polypeptide (GIP) receptor expression was responsible for unusual and rare cases of steroid-secreting adrenocortical tumors, the activity of which was triggered by food intake (4, 5). Other cases were recently reported where cortisol-secreting tumors responded in an unanticipated manner to such agents as ß2-adrenergic agonists or vasopressin (6, 7, 8).
Vasopressin has long been used as an investigative tool for Cushing’s syndrome with the concept that only patients with the Cushing’s disease would respond to the test with a cortisol rise secondarily induced by the direct action of vasopressin at the pituitary level (9, 10, 11, 12, 13). Hence, the apparently illegitimate response of cortisol in patients with a primary, ACTH-independent, adrenocortical tumor.
Vasopressin is classically associated with vasoconstriction, antidiuretic, and ACTH-releasing activities. Vasopressin is also involved in metabolic actions and in cell growth and differentiation. Three types of vasopressin receptors have been characterized and recently cloned [V1 (or V1a), V2, and V3 (or V1b)] (14, 15, 16, 17). The presence of V1 receptors in adrenal gland (18) has raised the possibility that vasopressin can directly stimulate adrenal steroidogenesis in both physiological and pathological conditions (19, 20). Recent studies have shown that vasopressin stimulated cortisol and aldosterone secretion in vitro from normal human adrenal fragments and cultured cells via activation of V1 receptors (21, 22). These data suggested that vasopressin receptor expression (at least of the V1 type) in the adrenal cortex was not an illegitimate phenomenon.
We hypothesized that an apparently aberrant vasopressin responsiveness of steroid-secreting adrenocortical tumors could result either from overexpression of the V1 receptor or from the illegitimate expression of other vasopressin receptor subtypes. Analysis of the various vasopressin receptors subtypes was performed by RT-PCR competition-based quantitation and by in situ hybridization for the V1 type. We show that a minority of vasopressin-responsive adrenocortical tumors express large amounts of the V1 vasopressin receptor.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Retrospective study of vasopressin responsiveness in a series of patients with Cushing’s syndrome. We retrospectively examined the plasma cortisol and ACTH responses of 116 patients submitted to a vasopressin stimulation test (see below) in the last 24 yr at our institution. The test was performed at the initial work-up of the patients, who were all hypercortisolic (increased urinary cortisol excretion and/or lack of suppression under the low dose dexamethasone suppression test). All patients with adrenocortical tumors (n = 26) had undetectable and/or unresponsive ACTH plasma levels (<5 pg/mL). All patients with Cushing’s disease (n = 90) had typical laboratory findings and/or proven corticotroph adenomas after pituitary surgery.
The vasopressin stimulation test was performed as previously described (10); 10 IU vasopressin (lysine vasopressin, Ciba-Geigy, Lausanne, Switzerland) were administered im at 0800 h, and blood was drawn at 0, 15, 30, 45, and 60 min. The response to vasopressin was measured by substracting the cortisol value at 0 min from the highest value between 15–60 min. A positive response was arbitrarily defined as equal to or higher than 30 ng/mL.
Tissue studies in recently operated patients with adrenocortical tumors. Tissue specimens were obtained from 39 patients with adrenocortical tumors (20 adenomas and 19 carcinomas). The status of each tumor was further assessed by measuring the IGF-II messenger ribonucleic acid (mRNA) content (23). As expected, all adenomas (except 1) had normal contents, whereas most carcinomas (15 of 19) had elevated values (data not shown).
Six patients had undergone a vasopressin stimulation test; there were four responders (three adenomas and and one carcinoma) and two nonresponders (one adenoma and one carcinoma).
Specimens of normal adrenocortical tissue were obtained by careful dissection of glands in three patients who had undergone unilateral adrenalectomy for nonsecreting tumors.
The tissues at the time of surgery were immediately frozen in liquid nitrogen and stored at -80 C until nucleic acid extraction. The pathologist dissected the tumor and provided a sample of fresh, homogeneous, nonnecrotic tumor tissue. Large blood vessels and fibrotic areas were excluded during the preparation of each specimen.
RNA extraction and RT-PCR analysis
Total RNA was isolated by ultracentrifugation in guanidinium isothiocyanate-CsCl followed by treatment with ribonuclease-free deoxyribonuclease I. RT-PCR was performed according to standard protocols. Briefly, random primed complementary DNA (cDNA) were synthesized from 1 µg total RNA with 200 U Moloney murine leukemia virus reverse transcriptase (BRL, Gaithersburg, MD), and 10% of the cDNA reaction was directly used for each PCR. The same cDNA was used for PCR amplification of glyceraldehyde-3-phosphate dehydrogenase (G3PDH).
Four sets of specific oligodeoxynucleotides were designed: V1, 5'-CGGCTTCATCTGCTACAACATC-3 and 5'-CGAGTCCTTCCACATACCCGT-3'; V2, 5'-GCTTGGGCCTTCTCGCTCCTT-3 and 5'-GCACAAAGGGCGCCCCTTCCA-3'; V3, 5'-ACCCCCACAGCAGGCAAGG-3 and 5'-TCTCGGGTCAGCAGCATCAA-3'; and G3PDH, 5'-ATCCCATCACCATCTTCCAG and AGGGATGATGTTCTGGAGA-GC-3'.
To ensure complementary DNA-specific amplification, primers were chosen
either flanking an intron [V1 and V3 (personal data)] or overlapping
the junction of two exons (for V2; Fig. 1a
).
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). The nature of the fragments was
also confirmed by enzyme restriction analysis.
Quantitative/competitive RT-PCR for the V1 mRNA (Fig. 1, b and
c)
Briefly, mutated V1 cDNA was generated by insertion of 80 bp
(derived from pUC18) within a region of the V1 gene comprised between
the specific PCR primers used. Corresponding RNA was synthesized using
a RNA transcription kit. The concentration of mutant transcript was
determined by measuring adsorbance at 260 nm, and quality was checked
by agarose gel electrophoresis. In a first series of reactions, fixed
amounts of tumoral RNA were mixed with increasing amounts of synthetic
RNA ranging from 103-107 copies. After 27
cycles of amplification (45 s at 94 C, 45 s at 60 C, and 1 min at
72 C) and a final step of 10 min at 72 C, 40% of the PCR products were
separated on ethidium bromide-stained 3% agarose gel. A second series
of reactions was then performed with a narrowed range of mutant RNA
dilutions, spaced by 4-fold only, to allow for precise RNA copy number
determination for each individual tumor. After agarose gel separation
and ethidium bromide staining, the products were quantified under UV
light using NIH Image software. The logarithm of the ratio of band
intensities within each lane was plotted against the logarithm of the
copy number of synthetic RNA added per reaction. This function was
linear. The quantity of target message was determined where the ratio
of synthetic/authentic V1 band intensities was equal to 1 (Fig. 1c).
Results are presented in arbitrary units (1U corresponding to 1 copy V1
mRNA per 0.1 µg total RNA).
The two groups (adenomas and carcinomas) were compared by one-way ANOVA and unpaired Student’s test. Statistics were performed after logarithmic transformation of the data.
In situ hybridization
Normal and pathological tissues embedded in paraffin were used for in situ hybridization. Tissues were fixed in 4% buffered paraformaldehyde immediately after removal from the patient. After 24 h in the fixative solution, tissues were washed in 70% ethanol, dehydrated, and embedded in paraffin. Sections (7 µm) were cut and mounted on silanated histological slides.
The full-length cDNA (2.2 kilobases) of the V1 receptor was subcloned in pBluescript KS+ at the EcoRI site. After linearization by XbaI and SalI, the V1 receptor cDNA was transcribed by T3 or T7 RNA polymerases (Boehringer Mannheim, Mannheim, Germany) in the presence of [35S]UTP (Amersham) to generate, respectively, the antisense and sense probes. The detailed protocol for in situ hybridization has been recently published (24).
Perifusion experiments
The perifusion system technique was previously described (25). Briefly, a fragment of the tumor obtained at surgery was immersed in 200 mL DMEM and rapidly transported to the laboratory. The tumor tissue was diced into small pieces (1–2 mm3), rinsed three times with fresh medium (DMEM), mixed with Bio-Gel P2 (Bio-Rad, Richmond, CA) and transferred into polystyrene columns delimited by two Teflon pestles. The perifusion chambers were supplied with DMEM at constant flow rate (260 µL/min) at 37 C and pH 7.4. The perifusion medium was continuously gassed with a 95% O2-5% CO2 mixture. The tumor slices were allowed to stabilize for 2 h before any substance was administered. Test substances were dissolved in gassed DMEM and infused into columns at the same flow rate as DMEM alone. Effluent fractions were collected at 5-min intervals and kept at -20 C until assay. Vasopressin and [d(CH2)5,Tyr(OMe)2]arginine vasopressin ([d(CH2)5,Tyr(OMe)2]AVP) were purchased from Sigma Chemical Co. (St. Louis, MO). DMEM was supplied by Life Technologies (Grand Island, NY).
Cortisol concentrations were determined in all fractions collected by RIA using antisera developed in our laboratory (25). The antibodies showed significant cross-reaction with 11-deoxycortisol (5%), but the cross-reactivities were much lower with corticosterone and 11-deoxycorticosterone (0.28% and 0.10%, respectively). The antibodies exhibited very low cross-reaction (<0.01%) with all other steroids tested. The assay was sensitive enough to detect 1 pg cortisol. The intra- and interassay coefficients of variation were 2% and 8%, respectively.
Plasma assays
ACTH was measured by a highly specific immunoradiometric assay with a detection limit of 5 pg/mL (ELSA ACTH, CIS-Bio International, Gif-sur-Yvette, France), and plasma cortisol levels were measured using a competitive binding assay as previously described (26).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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)
Sixty-seven of the 90 patients with Cushing’s disease (76%) had
a positive (
30 ng/mL) cortisol response to vasopressin stimulation.
Seven of the 26 patients with Cushing’s syndrome due to a primary
cortisol-secreting adrenocortical tumor (27%) had a positive cortisol
response to vasopressin stimulation. This response was ACTH
independent; in fact, plasma ACTH levels were undetectable and remained
unresponsive (<5 pg/mL). Among the 7 responsive adrenocortical tumors,
there were 5 adenomas and 2 adrenocortical carcinomas.
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RT-PCR detection of vasopressin receptors in tissue RNA. Expression of the V1 receptor gene was easily detected (20 cycles PCR), giving the expected signal of 505 bp in all normal adrenal glands and all tumoral tissues. The expected 439-bp fragment was amplified with the V2 primers in most adrenal samples and in 1 human kidney that was used as a positive control (data not shown). However, the amount of V2 RT-PCR product appeared considerably lower than that of V1 RT-PCR product, as 36 cycles of PCR were needed. The signals of V1 and V2 were not detected without the RT reaction. Thus, normal adrenal gland and most adrenal tumors express both V1 and V2 receptors, at least at the mRNA level. In contrast, RT-PCR V3 signal was absent in all tissues, whether normal or tumoral, even after 38 cycles.
Quantitative/competitive RT-PCR of V1 mRNA (Fig. 3). RT-PCR competition-based
quantitation was performed for V1 mRNA to further validate the results
of comparative RT-PCR. Mean V1 mRNA was significantly decreased in
carcinomas compared to adenomas (2.2 x 103vs. 1.0 x 104; P <
0.01). However, the levels of V1 gene expression showed a wide range of
variation in both tumor groups.
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Effect of vasopressin on cortisol secretion by tumor tissue in
vitro (Fig. 4). The effect of
vasopressin on cortisol production was directly studied in
vitro on perifused adrenocortical tumor fragments of a patient
whose adenoma had high levels of V1 mRNA (5.0 x 104)
and who had a brisk cortisol response to vasopressin in vivo
(Fig. 4). Exposure of tumor tissue to vasopressin (10-7
mol/L) induced cortisol secretion that reached a maximum relative
increase over baseline (202 ± 27%) within 30 min. To investigate
the type of receptor involved in the stimulatory action of vasopressin,
tumor fragments were exposed to the nonapeptide in the presence of the
[d(CH2)5,Tyr(Ome)2]AVP, a
selective V1 vasopressin receptor antagonist. As shown in Fig. 4
vasopressin-evoked cortisol secretion was totally abolished by
[d(CH2)5,Tyr(Ome)2]AVP.
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).
In situ hybridization was performed to further characterize
the cell type(s) in adrenal gland that expressed the V1 receptor. The
results showed that V1 mRNA was present in the normal adrenal cortex,
mainly in the compact cells of the zona reticularis and less in the
zona glomerulosa and fasciculata. In situ hybridization
demonstrated a very high intensity signal in tumoral samples of two
patients who had high tissue contents in V1 mRNA by RT-PCR [9.5
x 103 in one adenoma (Fig. 5D); 3.3 x
104 in one carcinoma (Fig. 5F)]. The specific V1
expression was localized in adrenocortical tumoral cells, particularly
in compact cells with a nonhomogeneous pattern in the same tumor
(Fig. 5, D and F).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In contradiction with a long accepted dogma, we and others have described cases of vasopressin-responsive adrenocortical tumors in patients with ACTH-independent hypercortisolism (6, 7, 8). We show here that it occurs in a minority of such tumors (27%), which can be either benign or malignant. This figure is less than that reported by another group (7), who found 100% responders under a similar stimulatory test. In our retrospective study we were careful to check that all of our patients were hypercortisolic and had no ACTH response. We arbitrarily chose 30 ng/mL as a significant increase; a recent study by Dickstein et al. (13) showed that a positive cortisol response to vasopressin (arbitrarily defined as a 20% relative increase) was present in 74% of patients with Cushing’s disease. Using their criteria, our figures would not change (26% for adrenocortical tumors and 76% for Cushing’s diseases), giving an almost identical rate of responders for the group of patients with Cushing’s disease as in Dickstein’s study (13).
It is now well established that vasopressin can directly stimulate steroidogenesis in normal animal and human adrenocortical cells via the activation of V1 receptors (19, 20, 21, 22, 31). We hypothesized that the likelihood of a tumor being responsive to vasopressin would be directly correlated with the level of expression of a eutopic V1 receptor in the tumor. We indeed showed that only those patients with high V1 mRNA levels were responsive to vasopressin in vivo. In general, higher values were found in benign tumors, but an occasional carcinoma had both extremely high tumor V1 mRNA content and a brisk cortisol response to vasopressin in vivo.
Because V1 receptor gene expression occurs in many different cell types, including in vessels, it was necessary to demonstrate its presence in adrenocortical cells. Two studies of human adrenocortical tumors have shown a direct action of vasopressin in vitro on cortisol secretion and on intracytosolic calcium movements (8, 22). We show here the direct action of vasopressin on perifused fragments from an adenoma responsible for Cushing’s syndrome and show its blockade by a specific V1 antagonist.
Using in situ hybridization, we show the presence of V1 mRNA
directly in human adrenocortical cells and reveal its nonhomogeneous
pattern of distribution. In the normal adrenal cortex, a V1 mRNA signal
is present in all three zonas, yet it shows a preferential association
with the compact cells of the zona reticularis. This cell type is
supposedly responsible for preferential androgens production and may
participate in adrenocortical cell regeneration. These features might
have some connection with the proposed growth action of vasopressin on
adrenal cortex (32). In situ V1 mRNA signals were easily
detected in some tumors selected for their high contents, as determined
by quantitative RT-PCR. In the two studied tumors, the V1 mRNA signal
showed a highly heterogeneous pattern, but in both cases, it was absent
in clear cells and appeared specifically associated with compact cells:
in the adenoma with a spotty pattern (Fig. 5D), and in the carcinoma in
large homogeneous nodules made of undifferentiated cells (Fig. 5F).
These in situ data also confirmed that V1 mRNA was present
in tumor cells and not in vessels.
We found no evidence for V3 gene expression in normal or tumoral adrenal cortex. In contrast, we found a definite, although very low, RT-PCR signal for the V2 receptor in the normal gland and in most tumors. A previous study showing the lack of effect of the V2-specific agonist 1 deamino-8-D-arginine vasopressin (desmopressin) on a vasopressin-responsive adrenocortical tumor suggests that this receptor type is not involved with steroid secretion (6). It is more likely that this slight signal reflects the presence of V2 receptors in tumor-associated vessels.
A spectrum of more or less spectacular endocrine abnormalities can be observed when structurally normal membrane receptors are overexpressed. Extreme overexpression of ß2-adrenergic receptors in the heart of transgenic mice leads to abnormal function even in the absence of ligand (33, 34); apparently "illegitimate" expression of GIP or ß2-adrenergic receptors in adrenocortical tumors leads to cortisol oversecretion when the "illegitimate" receptor is triggered by physiological ("legitimate") conditions such as food intake, postural changes, or even stress. At the end of this spectrum, we show here that eutopic (legitimate?) V1 gene expression can modulate the phenotype of steroid-secreting adrenocortical tumors, at least in response to pharmacological manipulations.
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Acknowledgments |
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Footnotes |
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1 This work was supported in part by INSERM CJF 9208, the INSERM
Réseau de Recherche Clinique Comète, the Fondation de
France, and the Plan Hospitalier de Recherche Clinique. 
2 Recipient of a fellowship from the Assciazone Italiana Ricerca sul
Cancro and a Poste Vert from INSERM.
Received August 4, 1997.
Revised February 18, 1998.
Accepted February 26, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, intron; 
,
coding region; 
, transmembrane domain) and the respective primers
(
), oligonuleotides for hybridization (
), and expected fragments
of RT-PCR amplification (
). b, Map and scheme for the construction
of the competitive V1 mRNA. Mutated V1 cDNA was generated by insertion
of 80 bp within a region of V1 gene amplified. Corresponding RNA was
then synthesized. c, Example of quantitative RT-PCR of V1 mRNA. The
upper panel shows the gel analysis of RT-PCR reactions
with a narrow range of mutant RNA amounts. The lower
band in the agarose gel represents amplified DNA fragment
derived from tissue sample V1 mRNA, and the upper band
represents the superimposition of two bands from the competitor mutated
V1 mRNA. At the equivalent point of signal intensities between the two
bands, the amounts of target V1 mRNA are the same as the copy number of
competitor. This point is calculated as follows: a, plotting the
logarithm of the ratio of mutant over patient DNA, expressed in
densitometric units, as a function of the logarithm of the copies of
mutant V1 mRNA present in the same reaction; and b, extrapolating the
value for x = 0 (3.87 in the present case).
; <5
pg/mL). In vitro response: in vitro
perifusion of the tumor fragments showing the brisk response to
vasopressin (10-7; left) and its blockade
by the specific V1 antagonist
[d(CH2)5,Tyr(OMe)2]AVP
(10-6; right). The patterns represent the
mean (±SEM) of three independent perifusions. Each
point is the mean of two consecutive 5-min fractions.
The basal cortisol release (100%) was calculated as the mean of the
fractions just preceding the administration of the secretagogue.