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(Hypertension. 1996;27:1153-1159.)
© 1996 American Heart Association, Inc.

Articles

Endothelin Adrenocortical Secretagogue Effect Is Mediated by the B Receptor in Rats

Anna S. Belloni; Gian Paolo Rossi; Paola G. Andreis; Giuliano Neri; Giovanna Albertin; Achille C. Pessina; Gastone G. Nussdorfer

From the Departments of Anatomy (A.S.B., P.G.A., G.N., G.G.N.) and Clinical Medicine (G.P.R., G.A., A.C.P.), School of Medicine, University of Padua (Italy).

Correspondence to Prof G.G. Nussdorfer, Department of Anatomy, Via Gabelli 65, I-35121 Padova, Italy.

	Abstract

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Abstract We investigated the gene expression and localization of endothelin-1 (ET-1) receptor subtypes ET_A and ET_B in the rat adrenal cortex as well as their involvement in the corticosteroid secretagogue effect of ET-1 in vitro. Reverse transcription–polymerase chain reaction with primers specific for ET_A and ET_B cDNAs demonstrated the expression of both receptor genes in homogenates of adrenocortical tissue. However, in isolated zona glomerulosa and zona fasciculata cells, only ET_B mRNA was detected. Autoradiographic examination of the selective displacement of ¹²⁵I–ET-1 binding by BQ-123 and BQ-788 (specific ligands for ET_A and ET_B, respectively) indicated that zona glomerulosa possesses both ET_A and ET_B, whereas zona fasciculata is exclusively provided with ET_B. ET-1 enhanced in a concentration-dependent manner aldosterone and corticosterone secretions of dispersed zona glomerulosa and zona fasciculata cells, respectively. The ET_B antagonist BQ-788 markedly reduced the secretory response of zona glomerulosa cells and completely suppressed that of zona fasciculata cells, whereas the ET_A antagonist BQ-123 was ineffective. These findings indicate that in the rat, the adrenocortical secretagogue action of ET-1 is mediated by the ET_B receptor subtype and that the ET_A receptor is not directly involved in such an effect.

Key Words: endothelin • receptors, endothelin • adrenergic beta-antagonists • adrenal cortex • steroids • gene expression

	Introduction

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The 21–amino acid peptide ET-1, originally isolated from porcine aortic endothelium,¹ exerts a potent vasoconstrictor and pressor activity (for review, see References 2 through 4^{2 3 4} ). Increasing evidence indicates that ET-1 enhances in vivo and in vitro basal and agonist-stimulated aldosterone secretion of adrenal ZG of several mammalian species,^{5 6 7 8 9 10 11} including the rat.^{12 13 14 15} Findings are also available that ET-1 stimulates basal cortisol and corticosterone secretion of isolated human and rat ZF cells.^{16 125}I–ET-1 binding in the whole adrenal cortex with a preferential localization in the ZG has also been demonstrated by autoradiography.^{17 18 19 20 21}

Pharmacological and in vitro binding studies, as well as molecular biology investigations, have identified two subtypes of ET-1 receptors, ET_A and ET_B, that have been shown to be mainly expressed in vascular smooth muscle and endothelial cells, respectively (for review, see References 22 and 23^{22 23} ). High-affinity specific ET_A and ET_B receptors have been described in the whole adrenal gland and in the ZG.^{5 24 25 26 27 28} Recently, the presence of both ET_A and ET_B in the ZG and of ET_B in the ZF of the human adrenal cortex has been autoradiographically demonstrated by the use of selective ligands.^{28 29} This was further confirmed with both ¹²⁵I–ET-1 displacement binding and gene expression experiments on homogenates of human adrenal cortex²⁸ but has not yet been confirmed in the rat. Furthermore, the studies of the specific involvement of ET_A and ET_B in the steroidogenic secretagogue effect of ET-1 were limited by the lack of specific antagonists for each receptor subtype.^{11 15 30}

Thus, our purpose was to investigate whether both ET_A and ET_B genes are expressed and translated into functional receptors in the rat adrenal cortex in both ZG and ZF cells and to identify the receptor subtype that mediates the adrenocortical secretagogue response to ET-1 in vitro.

	Methods

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Animals and Reagents
Male adult Wistar rats (200 to 220 g body weight) were purchased from Morini (Reggio Emilia, Italy) and maintained on a 12-hour light/dark cycle at 20±2°C. The protocol of the experiments described below was approved by the Animal Research Committee of the Padua University.

Moloney murine leukemia virus reverse transcriptase (MuLV-RT; GeneAmp RNA PCR core kit) and Taq polymerase (AmpliTaq) were purchased from Perkin-Elmer Cetus. ET-1 and BQ-123, a selective ET_A antagonist,³¹ were obtained from Peninsula Laboratories, and BQ-788, a potent and selective ET_B antagonist,³² from Neosystem Laboratories. ¹²⁵I–ET-1 (specific activity, 2000 Ci/mmol) was purchased from Amersham Laboratories, medium 199 from DIFCO, and human and bovine serum albumin from Sigma Chemical Co. Commercial radioimmunoassay kits for aldosterone and corticosterone were obtained from IRE-Sorin and Eurogenetix, respectively.

Preparation of Adrenal Specimens
Rats were decapitated and their adrenal gland excised and freed of pericapsular fat. Adrenals were immediately frozen in liquid nitrogen and stored at -195°C until used for nucleic acid extraction. Other adrenal glands were gently decapsulated to separate ZG from inner zones and then hemisected; decapsulated adrenal halves were enucleated for removal of medulla and adjacent zona reticularis. Dispersed capsular (ZG) and inner (ZF) adrenocortical cells were obtained by collagenase digestion and mechanical disaggregation.³³ Inner cell contamination in capsular cell preparations, as evaluated by phase microscopy, was always less than 7%.

RNA Preparation
Total RNA was extracted from tissues and cells after homogenization by the guanidinium isothiocyanate method. After isolation, RNA was checked for integrity by gel electrophoresis and UV absorbance as previously detailed²⁸ ; total RNA concentrations were then calculated by spectrophotometric measurements at a wavelength of 260 nm.

Polymerase Chain Reaction
For use in the PCR, total RNA was reverse transcribed to cDNA with random hexamers (2.5 µmol/L) and 5 U of cloned MuLV-RT, as previously described.²⁸ After incubation at 42°C for 15 minutes, the temperature was raised to 95°C for 5 minutes, and then reaction mixture tubes were quickly chilled on ice. For amplification of the resulting cDNA, 20 µL of the RT mixture was used. The sample volume was increased to 100 µL with a solution containing 50 mmol/L KCl, 10 mmol/L Tris (pH 8.3), 2 mmol/L MgCl₂, and 2.5 U Taq polymerase as well as 0.1 or 0.3 µmol/L of upstream and downstream primers for ET_A and ET_B, respectively. On the basis of the sequence of rat cDNA, the following primers, originally designed by Terada et al,³⁴ were used: For ET_A, the sense primer (bases 455 through 474) was 5'-GTGTTTAAGCTGTTGGCGGG-3' and the antisense (bases 1215 through 1234) was 5'-CGAGGTCATGAGGCTTTTGG-3'.^{34 35} For ET_B, the sense primer (bases 724 through 748) was 5'-AGCTGGTGCCCTTCATACAGAAGGC-3' and the antisense (bases 1621 through 1642) was 5'-TGCACACCTTTCCGCAAGCACG-3'.²⁷ For ET_A, the thermal profiles, used in a Delphi thermal cycler (Oracle Biosystem, MJ Research Inc) for a total of 38 cycles, differed from those published³⁴ only for the extension step at 72°C, which lasted for 1 minute. For ET_B, a denaturation step at 94°C for 1 minute, annealing step at 65°C for 1 minute, and extension step at 72°C for 1 minute for a total of 40 cycles were used.³⁴ Additional primers for the ET_A cDNA, designed with the OLIGO software (version 4.1, Medprobe), were as follows: sense, 5'-CCTTATCTACGTGGTCATTG-3' (bases 421 through 440), and antisense, 5'-GGTTCTGCTCCTGGTTCTTC-3' (bases 1264 through 1283).³⁵ Amplification was carried out for 40 cycles with a denaturation step at 94°C for 1 minute and annealing step at 56° or 58°C for 1 minute. An additional extension step at 72°C for 7 minutes was then carried out. To rule out the possibility of genomic DNA amplification, we performed PCR in some experiments without prior RT of the RNA.

Autoradiographic Studies
Adrenal cortexes from six rats were immediately frozen at -30°C by immersion in isopentane and stored at -80°C. Frozen 10- to 15-µm-thick sections were cut in a cryostat (Leitz 1720 Digital) at -20°C and processed according to Kuhar³⁶ and Palacios et al.³⁷ Sections were preincubated in 50 mmol/L Tris-HCl (pH 7.4) containing 0.01% bacitracin, 135 mmol/L NaCl, 10 mmol/L MgCl₂, 1 mmol/L tetrasodium EDTA, and 0.2% bovine serum albumin for 15 minutes at 20°C. ET-1 binding sites were labeled in vitro by incubation for 120 minutes with 100 pmol/L ¹²⁵I–ET-1; nonspecific binding was determined by addition of 100 nmol/L unlabeled ET-1. Selective ¹²⁵I–ET-1 binding to ET_A and ET_B was studied by addition of 100 nmol/L BQ-123 and BQ-788, respectively. The reaction was stopped by washing the samples three times in 50 mmol/L Tris-HCl buffer. After rinsing, the sections were rapidly dried, fixed in paraformaldehyde vapors at 80°C for 120 minutes, and coated with NTB2 nuclear emulsion (Eastman Kodak). Autoradiograms were exposed for 2 weeks at 4°C and then developed with undiluted Kodak D19 developer. They were stained with hematoxylin-eosin and observed and photographed with a Leitz Laborlux microscope. Three autoradiograms obtained from each adrenal were analyzed by computer-assisted densitometry with a camera-connected microscope and computer (Hantares-80) equipped with software specifically written for this purpose (Studio Casti Imaging). For each autoradiogram, 10 areas (approximately 25 000 µm² or 36 000 pixels) of ZG or ZF were analyzed.

Steroid Secretion Studies
Dispersed cells were obtained as described for the gene expression studies. The viability of isolated cells was checked by the trypan blue exclusion test and found to be higher than 92%. Six separate incubation experiments were performed, with cell suspensions used in each of them obtained from adrenal pairs of six rats. Dispersed cells were put in medium 199 and potassium-free Krebs-Ringer bicarbonate buffer with 2% glucose containing 5 mg/mL human serum albumin. Capsular and inner cells were incubated (3x10⁵ cells per milliliter) as follows: (1) increasing concentrations of ET-1 (from 10^-12 to 10^-6 mol/L), (2) 10^-9 mol/L ET-1 in the presence of increasing concentrations of BQ-123 or BQ-788 (from 10^-11 to 10^-6 mol/L), and (3) 10^-7 mol/L BQ-123 and 10^-7 mol/L BQ-788 in the presence or absence of 10^-9 mol/L ET-1. Incubations were carried out in a shaking bath at 37°C for 90 minutes in an atmosphere of 95% O₂ and 5% CO₂. At the end of the experiments, the incubation tubes were centrifuged at 4°C, and aldosterone and corticosterone were extracted from supernatants and purified by high-performance liquid chromatography.³⁸ Aldosterone and corticosterone concentrations were measured by radioimmunoassay with the following commercial kits: (1) aldosterone CTK2 (sensitivity: 5 pg/mL; cross-reactivity: aldosterone 100%, 17-iso-aldosterone and other steroids <0.1%) and (2) CORTX-RIA (sensitivity: 25 pg/mL; cross-reactivity: corticosterone and cortisol 100%, 11-deoxycorticosterone and progesterone 2%, and other steroids <0.001%). Intra-assay and interassay coefficients of variation were 5.5% and 7.2% for aldosterone and 7.3% and 8.6% for corticosterone, respectively.

Statistics
Data are expressed as mean±SE. Statistical comparison was done by ANOVA followed by Duncan's multiple range test.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Gene Expression
RT-PCR consistently allowed detection of ET_A and ET_B mRNAs in homogenates of all adrenal specimens examined. Examples of ethidium bromide–stained 1.5% agarose gels are shown in Fig 1. No amplification was seen in the control PCR containing either no cDNA (water) or total RNA without prior RT, thereby ruling out the possibility of false-positive results and of amplification of genomic DNA, respectively. As can be seen, amplified cDNA fragments of the expected size for the ET_B receptors were detected in both homogenates and adrenocortical ZG and ZF cells (Fig 1C). Multiple bands were obtained for the ET_A cDNA with the primers of Terada et al (Fig 1A).³⁴ We therefore investigated expression of the ET_A gene with another set of newly designed primers that confirmed the presence of ET_A mRNA in adrenocortical homogenates but not in isolated ZG and ZF cells (Fig 1B).

View larger version (26K): [in this window] [in a new window]   Figure 1. Ethidium bromide–stained 1.5% agarose gels show cDNA amplified with rat ETA- and ETB-specific primers. A, cDNA was amplified from homogenates of two different rat adrenal cortexes. Lane 1 was loaded with 200 ng of a size marker (Marker VIII, Boehringer Mannheim). The amplified fragments were of the expected sizes (919 bp for ETB and 780 bp for ETA). No amplification of water and RNA without prior RT, as a negative control, is also shown. B, cDNA was amplified from homogenate of the adrenal cortex (lane 2) and from dispersed rat ZG (lanes 3 and 4) and ZF (lanes 5 and 6) cells with newly designed rat ETA-specific primers (see "Methods"). Lane 1 was loaded with 200 ng of the size marker. The amplification product was of the expected size (863 bp). No amplification was detectable in lane 7, which was loaded with water and the PCR mixture but not template cDNA. C, cDNA was amplified from homogenate of a different rat adrenal cortex (lane 2) and from dispersed rat ZG (lanes 3 and 4) and ZF (lanes 5 and 6) cells with rat ETB-specific primers. Lane 1 was loaded with 200 ng of the size marker. The amplification product was of the expected size (919 bp). No amplification was detectable in lane 7, which was loaded with water and the PCR mixture but not template cDNA.

Autoradiography
¹²⁵I–ET-1 binding was intense in the ZG and moderate in the ZF. Adrenal capsule was not labeled, whereas extracapsular vessels displayed evident ¹²⁵I–ET-1 binding on both their muscular and endothelial components (Fig 2A). The addition of an excess of cold ET-1 displaced virtually all ¹²⁵I–ET-1 binding (Fig 2B). BQ-123 markedly attenuated labeling in ZG and completely eliminated it in the vascular tunica muscularis, without apparently affecting ¹²⁵I–ET-1 binding of ZF (Fig 2C). BQ-788 notably reduced labeling in ZG and virtually eliminated it in ZF; vascular smooth muscle cells were still labeled (Fig 2D). Quantitative densitometric data confirmed these qualitative descriptions for adrenocortical tissue (Fig 3).

View larger version (140K): [in this window] [in a new window]   Figure 2. Autoradiographs of frozen sections of rat adrenal cortex incubated with 125I–ET-1. A, Binding is more intense in ZG than ZF; extracapsular vessels (v) are also labeled. B, 125I–ET-1 binding is completely displaced by addition of cold ET-1. C, BQ-123 eliminates binding to the extracapsular vessels and markedly attenuates it in ZG; labeling of ZF is not changed. D, BQ-788 strongly decreases 125I–ET-1 binding in ZF and attenuates it in ZG, where it remains localized just beneath the capsule; the walls of extracapsular vessels are still labeled. c indicates gland capsule. Original magnification x110.

View larger version (44K): [in this window] [in a new window]   Figure 3. Evaluation by quantitative densitometry of 125I–ET-1 binding in rat adrenal cortex and of its displacement by cold ET-1 and selective ligands of ETA and ETB. Bars are mean±SE (n=6). *P<.01 vs total binding; AP<.01 vs ET-1–displaced binding.

Steroid Secretion
ET-1 raised aldosterone secretion of dispersed capsular cells in a concentration-dependent manner (Fig 4). The effect was already apparent at an ET-1 concentration of 10^-11 mol/L (65%) and reached its maximum at a concentration of 10^-9 and 10^-8 mol/L (2.6- and 2.9-fold, respectively). ET-1 also enhanced corticosterone output by dispersed inner cells, minimal and maximal effective concentrations being 10^-10 mol/L (70%) and 10^-9 mol/L (2.5-fold), respectively (Fig 4). BQ-123 did not affect the stimulatory effect of the maximally effective ET-1 concentration (Fig 5). Conversely, BQ-788 decreased in a concentration-dependent manner 10^-9 mol/L ET-1–stimulated output of both aldosterone by ZG cells (Fig 5A) and corticosterone by ZF cells (Fig 5B). In both cases, minimal and maximal effective concentrations of BQ-788 were 10^-9 mol/L (aldosterone, -22%; corticosterone, -40%) and 10^-7 mol/L (aldosterone, -41%; corticosterone, -70%). However, maximal BQ-788 concentration did abolish the corticosterone but not the aldosterone response to ET-1 (Fig 5); similar results were obtained after the simultaneous exposure to maximal effective concentrations of BQ-123 and BQ-788 (Fig 6).

View larger version (21K): [in this window] [in a new window]   Figure 4. Effect of ET-1 on aldosterone (ALDO) and corticosterone (B) secretion by dispersed rat ZG and ZF cells, respectively. Data are mean±SE (n=6). +P<.05, *P<.01 vs baseline.

View larger version (20K): [in this window] [in a new window]   Figure 5. Effect of selective antagonists of ETA and ETB receptor subtypes on secretory response to ET-1 of dispersed rat ZG (A) and ZF (B) cells. Data are mean±SE (n=6). ALDO indicates aldosterone; B, corticosterone. +P<.05, *P<.01 vs control group; aP<.05, AP<.01 vs baseline.

View larger version (22K): [in this window] [in a new window]   Figure 6. Effects of simultaneous exposure to 10-7 mol/L BQ-123 and BQ-788 on basal and ET-1–stimulated aldosterone (ALDO) and corticosterone (B) secretions of dispersed rat ZG (left) and ZF (right) cells. Data are mean±SE (n=6). *P<.01 vs respective control value; aP<.05 vs respective basal value; AP<.01 vs respective basal value.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study takes advantage of the identification and cloning of two rat endothelin receptor genes^{27 34} and the development of specific antagonists for each of them. The isopeptide-selective ET_A receptor, identified from a cDNA library derived from rat vascular smooth muscle cells,³⁴ preferentially binds ET-1 and is specifically antagonized by BQ-123.³¹ Its mRNA has been detected in the central nervous system as well as in the aorta, heart, bronchial smooth muscle cells, and pituitary gland.³⁹ At variance, the nonisopeptide-selective ET_B, originally identified from a rat lung cDNA library,²⁷ was found to bind ET-1, ET-2, and ET-3 with the same affinity and to be antagonized by BQ-788.³² It was detected on endothelial cells, where it can mediate the release of nitric oxide and prostacyclin, and also on vascular and nonvascular smooth muscle cells.^{22 27 39}

Our results show that these two endothelin receptor subtypes are expressed in the rat adrenal cortex as transcription and translation products. With the use of specific primers, we have been able to consistently amplify cDNA fragments of the expected size for both the ET_A and ET_B genes after RT from total RNA of adrenocortical homogenates. Of interest, multiple amplification products were evident for ET_A when amplification was carried out according to a published methodology.³⁴ The fact that analysis of the ET_A cDNA sequence with specific software revealed multiple (false) priming sequences for both the sense and antisense primers might account for this result. Alternatively, it is conceivable that the smaller, more intense PCR amplification band (Fig 1A and 1B) may correspond to a subtype of the ET_A receptor resulting from an alternative splicing, a finding that has been reported also in the human adrenal cortex.⁴⁰ However, this latter interpretation is not supported by our findings obtained with the use of newly designed primers, which revealed no false priming under low-stringency conditions for both the ET_A and ET_B cDNAs. With the use of these primers, we found the ET_A mRNA in homogenates of the adrenal cortex but were unable to detect its presence in isolated ZG and ZF cells (Fig 1B).

To obtain a more precise anatomic location of the receptor subtypes, we performed autoradiography with ¹²⁵I–ET-1 in the presence and absence of the specific ligands for ET_A and ET_B receptor subtypes, BQ-123 and BQ-788, respectively. These studies clearly show that rat ZG possesses both ET_A and ET_B, whereas ZF is exclusively provided with ET_B (Fig 2). However, as these studies were carried out at the light-microscopic level, our morphological findings did not allow us to conclusively ascertain whether ET-1 receptors are located on the parenchymal steroid-secreting cells or on the vascular components of the adrenal cortex (ie, ET_A on the smooth muscle cells of the arterial walls, and ET_B on the endothelial lining of the capillaries). The arrangement of the adrenal vascular supply is very peculiar (for review, see References 41 and 42^{41 42} ): Main arteries divide near the gland capsule and form a plexus, from which arterioles arise that penetrate into the outer ZG, where they open in the capillary network of the cortex that drains into the medullary veins. Only exceptionally, arterioles arising from the capsular plexus pass throughout the cortex, reaching directly the medulla. Thus, the presence of both arterioles and capillaries may well account for the presence of both ET_A and ET_B in the ZG and the lack of ET_A in the ZF, which is virtually deprived of arterioles (Fig 2). Our gene expression and steroid-secretion findings on dispersed ZG and ZF cells concur to rule out the exclusive vascular expression of the ET_B receptor subtype.

The results of our functional experiments demonstrate that ET-1 exerts a clear-cut direct secretagogue effect on both ZG and ZF dispersed adrenocortical cells (Fig 4). More importantly, they clarify the controversial issue of the receptor subtype, which mediates the aldosterone response to ET-1 stimulation. Previous in vitro experiments on calf adrenal ZG cells with different agonists pointed to the ET_B as a likely candidate,^{11 25} but other findings indicate that the ET_A antagonist BQ-123 may inhibit aldosterone secretion in this cell type.⁴³ Furthermore, the lack of specific and powerful agonists and antagonists for the ET_B receptor subtype has precluded definitive conclusions so far. Our data strongly indicate that the ET_B receptor subtype, which was autoradiographically localized in all the cortex, is involved in the direct secretagogue action of ET-1 on both ZG and ZF cells. In fact, BQ-788 was found to displace ¹²⁵I–ET-1 binding to ET_B and to inhibit both mineralocorticoid and glucocorticoid secretion of ZG and ZF cells, respectively. Of interest, we previously obtained nearly identical results (not shown) with IRL-1038, another ET_B antagonist,⁴⁴ which has been retracted because of inconsistent activity.⁴⁵ Our conclusion is supported also by the fact that in rats, sarafotoxin-S6c, a weak ET_B agonist,⁴⁶ was found to enhance aldosterone production both in vitro²⁵ and in vivo.¹⁵ Conversely, we found that BQ-123 displaced the ¹²⁵I–ET-1 binding to ET_A in the ZG but did not affect the secretory response to ET-1 of either ZG or ZF cells. Our findings that ET_A mRNA is not detectable in isolated ZG and ZF cells and that the ET_A receptor subtype is not directly involved in the steroidogenic secretagogue effect of ET-1 are also in keeping with the results of in vivo¹⁵ and in vitro³⁰ studies as well as with the findings that in rats, BQ-123 blocked the pressor effect of ET-1 but not the ET-1–induced increase in adrenal aldosterone content.¹⁵ It has to be acknowledged that our results do not clarify why BQ-788 (10^-7 mol/L) abolished the secretory response to ET-1 of ZF cells but only reduced (by about 70%) that of ZG cells. Functional evidence of the existence of a subtype of ET-1 receptors that is insensitive to both ET_A- and ET_B-specific ligands has been provided by several groups (for review, see References 3, 4, 21, and 47^{3 4 21 47} ). Therefore, further studies are ongoing to ascertain whether rat ZG cells may express this third type of ET-1 receptors, as our gene expression experiments suggest.

Another point that needs to be addressed concerns the functional role of ET_A in the rat adrenal ZG. It has been reported that ET-1 is a weak mitogen for cultured vascular muscle cells, fibroblasts, and glomerular mesangial cells but stimulates DNA and protein synthesis and cell proliferation in pulmonary artery (for review, see Reference 4⁴ ). Recently, evidence that this latter effect is mediated by the ET_A receptor subtype has been provided.⁴⁸ Our previous experiments showed that ET-1 can exert a mitogenic action on rat adrenal ZG but not ZF cells.⁴⁹ Since we have also shown that both ET_A and ET_B receptor subtypes are expressed and functionally detectable in the normal human ZG and in aldosterone-producing tumors,⁵⁰ the hypothesis that ET_A is involved in the maintenance (and stimulation) of the growth of normal and tumorous adrenocortical tissues can be put forward. However, the fact that the ET_A mRNA was not detected in isolated rat adrenocortical cells is not consistent with this hypothesis. An alternative possibility, awaiting further investigation, stems from the above-discussed probable localization of ET_A receptors in the ZG arterioles: ET-1 via these receptors could be involved in the regulation of adrenal blood flow, which is known to be at least in the rat an important modulator of steroid release (for review, see Reference 51⁵¹ ).

	Selected Abbreviations and Acronyms

 

ET-1, -2, -3 = endothelin-1, -2, -3 ETA, ETB = endothelin receptor subtype A, subtype B PCR = polymerase chain reaction RT = reverse transcription ZF = zona fasciculata ZG = zona glomerulosa

	Acknowledgments

 
This study was supported in part 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 CNR grant 94.00859.CT04.

Received October 19, 1995; first decision November 10, 1995; accepted January 22, 1996.

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