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(Hypertension. 1995;25:842-847.)
© 1995 American Heart Association, Inc.

Articles

Endothelin-1 and Its Receptors A and B in Human Aldosterone-Producing Adenomas

GianPaolo Rossi; Anna S. Belloni; Giovanna Albertin; Lucia Zanin; Maria Angela Biasolo; Gastone G. Nussdorfer; Giorgio Palù; Achille C. Pessina

From the Departments of Clinical Medicine (G.R., G.A., L.Z., A.C.P.), Microbiology (M.A.B., G.P.), and Anatomy (A.S.B., G.G.N.) of the University of Padova (Italy) Medical School.

Correspondence to GianPaolo Rossi, MD, FACC, Hypertension Unit, Clinica Medica 1, University Hospital, via Giustiniani, 2, 35126 Padova, Italy.

	Abstract

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Abstract Endothelin-1 stimulates aldosterone secretion by interacting with specific receptors. Accordingly, we wished to investigate endothelin-1, endothelin-A (ET_A) receptor, and endothelin-B (ET_B) receptor gene expression, localization, and properties in aldosterone-producing adenomas and in the normal human adrenal cortex. We carried out ¹²⁵I–endothelin-1 displacement studies with cold endothelin-1, endothelin-3, the specific ET_A antagonist BQ-123, and the specific ET_B weak agonist sarafotoxin 6 C and coanalyzed data with the nonlinear iterative curve-fitting program LIGAND. We also studied gene expression with reverse transcription–polymerase chain reaction with specific primers for endothelin-1, ET_A, and ET_B complementary DNA. Normal adrenal cortices from consenting kidney cancer patients (n=2) and aldosterone-producing adenomas (n=4) were studied; for the latter, surrounding normal cortex and kidney biopsy tissue served as controls. To further localize the receptor subtypes, tissue sections were studied by autoradiography in the presence and absence of 500 nmol/L BQ-123, 100 nmol/L sarafotoxin 6 C, and 1 µmol/L cold endothelin-1. In all tissues examined, endothelin-1, ET_A, and ET_B messenger RNAs were easily detected. However, in aldosterone-producing adenomas, both receptors' genes were expressed at a higher level than in the kidney. In aldosterone-producing adenomas (F=9.49, P<.01) as well as in the normal adrenal cortex (F=8.57, P<.01), but not in adrenocortical tissue surrounding aldosterone-producing adenomas (F=5.08, P=NS), the significantly best fitting of binding data was provided by a two-site model indicating the presence of two receptor subtypes with density (B_max) and affinity (K_d) similar to those previously found in other tissues. Autoradiography confirmed the presence of both ET_A and ET_B receptors on normal zona glomerulosa cells as well as on aldosterone-producing adenoma cells. Thus, the genes of endothelin-1 and of its receptors, ET_A and ET_B, are actively transcribed in the human adrenal cortex, and both receptor subtypes are translated into proteins in zona glomerulosa and aldosterone-producing adenoma cells. These data are consistent with an autocrine-paracrine role of endothelin-1 in the regulation of aldosterone secretion, both under normal conditions and in aldosterone-producing adenomas.

Key Words: hypertension, endocrine • aldosterone • adrenal glands • endothelins • receptors, endothelin

	Introduction

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The very potent 21–amino acid vasoconstrictor peptide endothelin-1 (ET-1) has recently been shown to stimulate aldosterone secretion, both in vitro and in vivo,^{1 2 3 4 5 6 7} and to enhance corticotropin- and angiotensin II–stimulated aldosterone secretion.^{8 9} Because endothelial damage can turn on ET-1 synthesis and secretion, the peptide is a likely mediator of the hyperaldosteronism of several conditions in which enhanced ET-1 synthesis and endothelial damage coexist, including liver cirrhosis, congestive heart failure, preeclampsia, liver transplantation, endotoxemic shock, and primary and malignant hypertension.^{10 11 12 13 14 15 16 17 18} Of interest, the chronic infusion of ET-1 was found to raise plasma aldosterone concentration in rats and cause a notable hypertrophy of the zona glomerulosa (ZG) cells; furthermore, ZG cells isolated from the infused animals exhibited an enhanced basal production of aldosterone.^{19 20} These findings raise the hypothesis that ET-1 may be implicated in causing cell growth and enhanced aldosterone secretion in primary aldosteronism, a condition in which no known stimulus of ZG cell growth and enhanced aldosterone secretion has yet been identified.²¹

The physiological effects of ET-1 appear to be mediated by two different ET-1–specific receptors, ET_A and ET_B, which have been pharmacologically characterized.²² Autoradiographic evidence of ET-1–specific binding to the rat, porcine, and human adrenal cortex, as well as to cultured calf adrenal ZG cells, has been reported.^{2 23 24 25 26} Northern blot analysis demonstrated the expression of ET_A and ET_B receptors in homogenates of rat adrenals; furthermore, in hybridization experiments in situ, localization of the messenger RNA (mRNA) of ET_A to the corticomedullary junction was observed, whereas ET_B was found to be diffusely distributed throughout the adrenal cortex and medulla.²⁷ However, in another study, the ET_B receptor was detected immunochemically on the endothelial lining of capillaries around the ZG and in the zona fasciculata but not on the ZG steroidogenic cells of bovine adrenals.²⁸ By taking advantage of the recent development of the specific ET_A antagonist BQ-123 and of the ET_B weak agonist sarafotoxin 6 C, we have recently provided evidence of the existence of both ET_A and ET_B receptors in the normal human ZG.²⁹ This finding was further confirmed by autoradiography and gene expression studies, both with a reverse transcription–polymerase chain reaction (RT-PCR) on normal adrenal cortices and aldosterone-producing adenoma (APA) tissue³⁰ and with Northern blot analysis of human adrenal glands of three patients with APA.³¹ However, the finding of ET-1 receptor expression on homogenates of APA tissue was not consistent with the functional observation that ET-1 stimulated in a dose-dependent fashion the secretion of aldosterone in vitro from normal adrenal cortices and from the cortex surrounding APA, but not from the tumors in patients with primary aldosteronism.³²

Thus, our purpose was to investigate whether and where ET_A and ET_B receptors are expressed in APA and to assess their anatomic distribution and their binding properties compared with the normal adrenal cortex.

	Methods

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Preparation of Adrenal Tissues
We studied histologically normal adrenal glands, obtained at surgery from consenting patients undergoing unilateral nephrectomy for kidney cancer (n=2), and adenomatous adrenal glands of patients with Conn's disease (n=4). For the latter tissues, the surrounding normal cortex and renal cortex biopsy tissues served as controls for the gene expression studies. After excision, tissues were immediately frozen in liquid nitrogen and stored at -195°C until they were used for ET-1 binding studies, nucleic acid extraction, and autoradiography. The protocol followed our institutional guidelines for the use of human tissue.

Binding Study
After homogenization, centrifugation, and resuspension of the cells in a Tris-HCl buffer, protein concentration was measured with a modified Lowry method. Membrane suspensions (15 to 25 µg protein) were then incubated with 25 pmol/L ¹²⁵I–ET-1 (Amersham Laboratories; specific activity, 2000 Ci/mmol) in the absence and presence of increasing concentrations of unlabeled ET-1, ET-3, sarafotoxin 6 C³³ (Sigma Aldrich), and BQ-123 (Peninsula Laboratories Inc), as already reported. The binding experiments were analyzed by the nonlinear iterative curve-fitting program LIGAND^{34 35} (Ligand, Biosoft) to establish the model that provided the significantly best fit (P<.05) by use of the F test and to obtain final parameter estimates of the dissociation constant (K_d) and receptor density (B_max) values.

Gene Expression Studies
Total RNA was checked for integrity by gel electrophoresis and ultraviolet absorbance as reported.²⁸ After reverse transcription, PCR amplification (GeneAmp RNA PCR Kit, Perkin-Elmer) was carried out, as previously reported in detail.³⁰ To rule out the possibility of genomic DNA amplification, in some experiments the PCR was performed without prior reverse transcription of the RNA. The digoxigenin-labeled amplification products underwent size-fractionation on 1.5% agarose gel electrophoresis stained with ethidium bromide, followed by Southern blotting onto a nylon membrane, ultraviolet cross-linking (Stratagene UV-Crosslinker 1800, Stratagene-Duotech), and detection by chemiluminescence (DIG, Boehringer Mannheim), as previously reported.³⁰

Autoradiography
Frozen 10- to 15-µm sections of APA and normal adrenal cortices, immediately frozen in isopentane cooled in liquid nitrogen in the operating room, were cut in a cryostat (Leitz 1720 Digital) at -20°C and processed as reported previously.^{29 30} After preincubation, sections were labeled in vitro by incubation for 120 minutes with 100 pmol/L ¹²⁵I–ET-1 at room temperature; nonspecific binding was determined by adding 1 µmol/L cold ET-1. Selective displacement of ¹²⁵I–ET-1 was studied by adding 500 nmol/L BQ-123 or 100 nmol/L sarafotoxin 6 C. Reaction was terminated by washing of the samples three times in 50 mmol/L Tris-HCl buffer. After being rinsed in distilled water, the sections were rapidly dried, fixed in paraformaldehyde vapors at 80°C for 120 minutes, and then coated with NTB2 Kodak Nuclear emulsion (Eastman Kodak). The autoradiograms were exposed for 2 weeks at 4°C and were then developed with undiluted D19 Kodak developer. They were stained with hematoxylin-eosin and observed and photographed with a Leitz Laborlux microscope.

Statistical Analysis
Results are reported as mean±SEM. Comparison between groups was performed by Student's t test for unpaired data and the Mann-Whitney nonparametric test. Statistical analysis was performed with the SPSS/PC+ statistical package (SPSS Inc).

	Results

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¹²⁵I–ET-1 Displacement Binding
Cold ET-1 was the most potent in displacing ¹²⁵I–ET-1, followed by sarafotoxin 6 C, ET-3, and BQ-123. In normal and APA tissue, both sarafotoxin 6 C and BQ-123 produced biphasic competition binding curves, suggesting the presence of two binding sites. Analysis of the saturation and displacement experiments showed Hill and pseudo-Hill coefficient values less than unity for all ligands, suggesting the presence of multiple binding sites. Further analysis revealed that the best fit was provided by a two-site model, as shown by the significant values of the F ratio³⁴ both in normal adrenal cortices and in APA tissue but not in the normal adrenal cortex surrounding the tumors (Table). The estimated B_max values were similar in APA and normal cortices and were within the ranges reported for human tissues³⁵ ; the estimated K_d values were similar to those measured in vitro in cells transfected with human ET_A and ET_B complementary DNA³⁶ but were higher in normal cortices than in APA.

View this table: [in this window] [in a new window]   Table 1. F Ratio and Significance of the Two-Site Compared With the One-Site Model Fitting and Density (Bmax) Values for Endothelin-1

PCR Results
The RT-PCR consistently allowed detection of ET-1, ET_A, and ET_B mRNA in all adrenal specimens examined. An example of an ethidium bromide–stained 1.5% agarose gel is shown in Fig 1. As can be seen, amplified complementary DNA fragments of the expected size for both the ET_A and the ET_B receptors and for the control ß-actin gene were easily detected in the normal adrenal cortex and in the APA tissue. In the latter tissue, a notable difference is evident in the expression of the ET_A and ET_B receptors between both normal and APA tissue and the renal cortex, despite no evident difference in the expression of the ß-actin gene.

View larger version (60K): [in this window] [in a new window]   Figure 1. Photograph of ethidium bromide–stained agarose gel showing complementary DNA amplified with endothelin-1 (ET-1)–specific, endothelin-A (ETA)–specific, and ETB-specific primers from an aldosterone-producing adenoma, the surrounding histologically normal adrenal cortex, and kidney tissue from a patient with Conn's disease. Amplification of the ß-actin complementary DNA, as a positive control, and lack of amplification with no DNA template (water), as a negative control, are also shown. Amplification of a product of 442 bp with ET-1–specific primers from a cloned ET-1 construct (pGEM-3-ET-1) is also shown.

Autoradiography Findings
Specific ¹²⁵I–ET-1 binding was evident in all adrenals examined, both in the normal cortices and in the cortex surrounding tumors (not shown). In the tumors, it was mainly located in the capillaries and arterioles running among tumor cells. A more intense labeling of areas made of compact ZG-like cells than of areas of larger (lipid-laden) cells was observed (Figs 2 and 3A). The addition of an excess of cold ET-1 virtually displaced all ¹²⁵I–ET-1 binding (Figs 2 and 3B). BQ-123 completely eliminated labeling in the vascular tunica muscularis (Fig 2C and 2E), without apparently affecting ¹²⁵I–ET-1 binding of areas of compact tumor ZG-like cells and of capillary endothelium (Fig 2C and 2E). However, binding to larger clear zona fasciculata–type tumor cells was not affected (Fig 3C and 3E). Sarafotoxin 6 C determined either a moderate or a marked attenuation of labeling of compact ZG-like tumor cells (Fig 2D and 2F) and of light zona fasciculata–type tumor cells (Fig 3D and 3F), respectively; in addition, it completely displaced binding to endothelium, while tunica muscularis remained well labeled (Figs 2D, 2F, 3D, and 3F).

View larger version (320K): [in this window] [in a new window]   Figure 2. Autoradiographs of frozen sections of a human adrenal aldosterone-producing adenoma, made of compact glomerulosa-like cells, incubated with 125I–endothelin-1 (125I–ET-1) (100 pmol/L). A, Binding is well distributed but is more intense to the capillaries among the tumor cells. Weaker binding is evident in an area with light (lipid-rich) larger cells among capillaries on the right of the section. B, 125I–ET-1 is completely displaced by the addition of 1 µmol/L cold ET-1. C, BQ-123 completely displaces 125I–ET-1 binding to the tunica media of the arterioles but not to endothelium of the capillaries and does not attenuate binding to the tumor compact cells. D, Sarafotoxin 6 C attenuates 125I–ET-1 binding to the tumor compact cells, but the binding to the muscular wall of arterioles is not affected (original magnification x90). E and F, At higher magnification (original x350), the distribution of the silver granules on steroidogenic cells is evident both in the BQ-123–treated section (E, endothelin-B receptors) and in the sarafotoxin 6 C–treated (F, endothelin-A receptors) section. Arrows indicate capillaries in A and endothelium in C and E; asterisks, muscular wall of arterioles.

View larger version (203K): [in this window] [in a new window]   Figure 3. Autoradiographs of frozen sections of human adrenal aldosterone-producing adenoma, made of lipid-laden, clear, zona fasciculata–type cells, incubated with 125I–endothelin-1 (125I–ET-1) (100 pmol/L). A, Binding is mainly located on the capillaries among the islets of tumor cells. B, 125I–ET-1 is completely displaced by the addition of 1 µmol/L cold ET-1. C, BQ-123 does not affect labeling to tumor cells or to endothelium of the capillaries. D, Sarafotoxin 6 C markedly attenuates 125I–ET-1 binding to tumor cells and eliminates that to the endothelium of capillaries; the binding to the muscular wall of arterioles is not affected (original magnification x90). E and F, At higher magnification (original x350), the distribution of the silver granules on steroidogenic cells is still evident in the BQ-123–treated section (E, endothelin-B receptors), but it is much less evident in the sarafotoxin 6 C–treated section (F, endothelin-A receptors). Arrows indicate capillaries; asterisks, muscular wall of arterioles.

	Discussion

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Soon after their discovery, endothelins were deemed to play an important role in blood pressure regulation because of their multiple biological actions, including very potent and long-lasting vasoconstriction, mitogenesis, and stimulation of endothelium-derived relaxing factor, atrial natriuretic peptide, arginine vasopressin, and aldosterone release.^{1 4} In anesthetized dogs, the infusion of ET-1, aside from causing widespread hemodynamic effects, consistently increased plasma renin activity and aldosterone secretion.⁴ The latter seemed to be independent of the former, because it was also observed in vitro in dispersed ZG cells^{2 3 6} and even after blockade of angiotensin II formation.³⁶ It appears to involve a Ca²⁺-dependent mechanism^{7 37 38} and prostaglandin synthesis, as shown in perfused slices of frog adrenal gland.⁷ ET-1 was also found to potentiate the aldosterone response to angiotensin II and corticotropin.^{8 9 26 39} Of interest, corticotropin has been found to increase the release of ET-1 from the adrenals, thereby suggesting that ET-1 is a mediator of corticotropin-stimulated aldosterone secretion.⁴⁰ In vitro binding experiments on calf adrenal cultured glomerulosa cells have shown the presence of specific, saturable endothelin binding sites.^{2 23 24} Further studies on the same experimental model with the endothelin analogue sarafotoxin 6 B and with ET-3 have suggested the presence of a high-affinity and a low-affinity receptor.²⁶ In an immunochemical study, however, no staining of steroidogenic cells of ZG with an ET_B-specific antiserum was detected.²⁷ With the use of specific antagonists for both the ET_A and the ET_B receptor subtypes and of RT-PCR with specific primers for each of the human endothelin receptor genes, we have recently demonstrated that both receptors are expressed as transcription and translation products in the normal human adrenal cortex.³⁰ We were also able to localize them autoradiographically in the human ZG as well as to detect the ET_A and ET_B subtypes on the arteriolar tunica media and the endothelial lining of the sinusoids, respectively. This was in keeping with the results of Northern blot analysis and in situ hybridization studies of rat adrenal glands.²⁸ The reasons for the discrepancy between immunohistochemistry and molecular studies is likely to be due to the lower sensitivity of the former compared with the latter methodologies.²⁷

In this study, APA tissue was investigated in order for us to gain some insight into the possible autocrine-paracrine role of ET-1 in the excess aldosterone secretion and cell growth of this condition. The results of ¹²⁵I–ET-1 saturation binding displacement experiments with a specific ET_A antagonist and a specific ET_B ligand show that both receptor subtypes are detectable in APA tissue (Table). This is further confirmed by the results of gene expression studies, which in addition provide evidence of translation of the ET-1 gene in the tumor tissue (Fig 1). However, autoradiographic studies demonstrate a marked heterogeneity of expression of the two receptor subtypes in the different tumors as well as in different areas and structures of the same tumor (Fig 2). In fact, both ET_A and ET_B receptors were detected on compact tumor cells of one patient (Fig 2A through 2D), whereas only ET_B receptors were found in other tumors made of large lipid-rich cells (Fig 2E through 2H). This finding has obvious implications both for gene expression and binding experiments and for functional studies. Of interest, in five patients with primary aldosteronism, Zeng et al³² reported that ET-1 stimulated the secretion of aldosterone in vitro from normal adrenal cortex as well as from the cortex surrounding APA, but not from tumor slices; they therefore suggested that the latter might be lacking in ET-1 receptors. Our finding of a marked heterogeneity of receptor distribution might explain the discrepancy between those negative functional results and the demonstration of both receptors with ¹²⁵I–ET-1 displacement binding and gene expression studies. The latter are carried out on tissue homogenates and therefore provide averaged information on a bulk of different cells and structures of the tissue examined, including endothelium and arterioles' tunica media. In contrast, functional studies on tissue slices are likely to be critically dependent on the topographical location of the section being investigated. Thus, although the concept of a physiological role of ET-1 in the paracrine regulation of aldosterone secretion is supported by the finding that genes for ET-1 and its receptors, ET_A and ET_B, are consistently expressed and translated into protein in the normal human adrenal ZG cells, as well as in the cortex surrounding APA, the picture may be different in the context of APA tissue. The mRNAs for the ET_A and ET_B receptors are easily detectable, and these receptors can be functionally measured in this tissue. However, the marked heterogeneity of expression of the two receptor subtypes between different tumors and even among different areas of the same tumor suggests the possibility of an autocrine-paracrine downregulation of ET-1 receptors due to local activation of ET-1 synthesis; this hypothesis needs to be further explored.

	Acknowledgments

 
This study was supported by The Italian National Research Council (CNR)—Targeted Project "Prevention and Control of Disease Factors (FATMA)": Subproject 8, Contract No. 91.00.218 PF41 115.06.654, and by Regione Veneto, Giunta Regionale, Ricerca Finalizzata, Venezia, Italia.

	Footnotes

 
Presented, in part, at the 15th Scientific Meeting of the International Society of Hypertension, Melbourne, Australia, March 20-24, 1994.

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