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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 1 322-328
Copyright © 2002 by The Endocrine Society

Other Original Articles

Endothelin-1[1–31]: A Novel Autocrine-Paracrine Regulator of Human Adrenal Cortex Secretion and Growth

G. P. Rossi, P. G. Andreis, S. Colonna, G. Albertin, F. Aragona, A. S. Belloni and G. G. Nussdorfer

Departments of Clinical and Experimental Medicine (G.P.R., S.C.), Human Anatomy and Physiology, Section of Anatomy (P.G.A., G.A., A.S.B., G.G.N.), and Urology, School of Medicine (F.A.), University of Padua, 35121 Padua, Italy

Address all correspondence and requests for reprints to: Prof. G. G. Nussdorfer, Department of Human Anatomy and Physiology, Section of Anatomy, Via Gabelli 65, I-35121 Padova, Italy. E-mail: gastone.nusdorfer{at}unipd.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endothelin (ET)-1[1–21] stimulates steroid secretion and zona glomerulosa growth and is expressed in the human and rat adrenal cortex together with its receptor subtypes A and B (ETA and ETB). Although ET-1[1–21] is generated from bigET-1 by an ET-converting enzyme (ECE-1), there is evidence of an alternative chymase-mediated biosynthetic pathway leading to the production of an ET-1[1–31] peptide, the role of which in adrenal pathophysiology is largely unknown. Gene expression and immunohistochemical studies allowed localization of chymase in the normal human adrenal cortex. Sizable amounts, not only of ET-1[1–21] but also of ET-1[1–31], were found in the adrenal vein plasma of three patients. ET-1[1–21] and ET-1[1–31] elicited a clear-cut secretory response by dispersed human adrenocortical cells, ET-1[1–31] being significantly less potent than ET-1[1–21]. The secretagogue effect of ET-1[1–31] was abolished by the ETA receptor antagonist BQ-123 and was unaffected by the ETB receptor antagonist BQ-788. Because, in humans, the secretagogue effect of ET-1[1–21] involves both ETA and ETB receptors, the weaker action of ET-1[1–31] could be attributable to a selective ETA receptor activation. Two lines of evidence support this contention: 1) ET-1[1–31] was more effective than ET-1[1–21] in stimulating ETA-mediated cell proliferation of human adrenocortical cells cultured in vitro; and 2) autoradiography showed that a) ET-1[1–31] displaced in vitro [125I]ET-1[1–21] binding to the ETA, but not ETB receptors, in human internal thoracic artery rings; and b) BQ-123, but not BQ-788, eliminated [125I]ET-1[1–31] binding in the rat adrenal cortex.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOTHELIN (ET)-1 is the prototype of a family of 21-amino acid residue peptides, which act through two main receptor subtypes called ETA and ETB. ET-1[1–21] is generated from bigET-1 through cleavage at the Trp21-Val/Ile22 bond by a specific ET-converting enzyme (ECE)-1 (1). However, according to more recent evidence, bigET-1 may also be selectively cleaved in humans by a chymase at the Tyr31-Gly32 bond to produce ET-1[1–31], without any further degradation (2, 3). ET-1[1–31] has been found to reproduce many of the ETA receptor-mediated vascular effects of ET-1[1–21] (4), including contraction of porcine and rat aorta (5), raising of intracellular Ca2+ concentration in cultured human vascular smooth muscle cells (VSMC) (6, 7, 8), and stimulation of VSMC proliferation (9). Moreover, we have recently observed that, like ET-1[1–21], ET-1[1–31] enhances proliferation of cultured rat zona glomerulosa (ZG) cells, acting through the ETA receptor (10).

ET-1[1–21], which is locally produced in the adrenal glands, is deemed to act as an autocrine-paracrine regulator of adrenocortical cell function (11). Although the possibility exists that ET-1[1–31] is also synthesized in the adrenal cortex, the presence of chymase in the human adrenal cortex remains to be conclusively proven. Furthermore, studies of the effects of ET-1[1–31] on human adrenocortical secretion are not yet available. Therefore, we sought for the expression of chymase at both the gene and the protein level and investigated the in vitro effects of ET-1[1–31], compared with those of ET-1[1–21], on human adrenocortical cells.


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

ET-1[1–21] and the selective ETA- and ETB-receptor antagonists BQ-123 and BQ-788 (12) were purchased from Neosystem Laboratories (Strasbourg, France); and ET-1[1–31], from Peptide Institute (Osaka, Japan). [125I]ET-1[1–21] and [125I]ET-1[1–31] were provided by Amersham Pharmacia Biotech (Aylesbury, UK) Medium 199 was obtained from Difco (Detroit, MI); and the ECE-1 inhibitor phosphoramidone (13), FBS, human serum albumin, BSA, 5-bromo-2'-deoxyuridine (BrdU), and other laboratory reagents were provided by Sigma (St. Louis, MO).

Dispersed human adrenocortical cells

Adrenal glands were obtained from 10 consenting patients undergoing unilateral nephrectomy with ipsilateral adrenalectomy for renal cancer. The experimental protocol was approved by the local ethics committees. The tails of the human adrenals, which lack of medullary chromaffin tissue (14), were collected. The gland capsule was stripped to separate ZG, and dispersed ZG and zona fasciculata-reticularis (ZF/R) cells were obtained by collagenase digestion and mechanical disaggregation (14).

RT-PCR

Total RNA was isolated by TRIzol reagent (Life Technologies, Inc., Milan, Italy) from human adrenal glands, left ventricular myocardium, and internal thoracic artery. For use in the PCR, total RNA was reversely transcribed to cDNA. One microgram of total RNA was dissolved in 10 µl of a mixture containing (final concentration) 1 mM deoxy-ATP, deoxy-GTP, thymidine 5'-triphosphate, and deoxy-CTP; 1 U/µl ribonuclease inhibitor (PE Applied Biosystems, Foster City, CA); 2.5 µM random hexamers; 50 mM KCl; 10 mM Tris-HCl (pH 8.3); 5 mM MgCl2; and 2.5 U cloned Moloney Murine Leukemia Virus RT (M-MuLV-RT; PE Applied Biosystems). After incubation at 42 C for 15 min, temperature was raised to 99 C for 5 min and then reaction mixture tubes were quickly chilled on ice. For amplification of the resulting cDNA, the sample volume was increased to 50 µl with a solution containing 50 mM/L KCl, 10 mM Tris (pH 8.3), 2 mM MgCl2, and 0.1 µM up- and downstream primers, as well as 1 U Taq polymerase (AmpliTaq Gold DNA, PE Applied Biosystems). The thermal profile and the primers were selected with the software Oligo. In a thermal cycler (Delphi 1000, Oracle Biosystems, MJ Research Inc., Watertown, MA), we used a denaturation step at 95 C for 1 min, annealing at 60 C for 1 min, and extension step at 72 C for 1 min for a total of 38 cycles. An additional extension step at 72 C for 7 min was then carried out. Both the 5' and 3' primers used for human chymase (CMA1) mRNA consisted of 21 bases corresponding to sequences CMA1–85-5', sense (5'-AAG CCA CAT TCC CGC CCC TAC AT-3'), and CMA1–631-3', antisense (5'-CAC CCC AGC ACA CAG AAG AGG-3') (15). The 5' and 3' primers used for GAPDH mRNA consisted of 20 bases corresponding to sequences 130–149 (5'-CCC TTC ATT GAC CTC AAC TA-3') and 695–714 (5'-GCC AGT GAG CTT CCC GTT CA-3') (16). To rule out the possibility of amplifying genomic DNA, one PCR was carried out without prior reverse transcription (RT) of the RNA. As positive controls, RNA (extracted from a specimen of human myocardium) and internal thoracic artery, two tissues that are known to express chymase, were used. Detection of the PCR amplification products was carried out by size-fractionation on 2% agarose gel electrophoresis. The amplification products were of the expected size, which was 552 bp and 585 bp for human chymase and for GAPDH, respectively. Specificity of the amplification products of chymase and GAPDH was further verified by melting curve analysis in a fluorescent thermal cycler (Icycler, Bio-Rad Laboratories, Inc., Milano, Italy), using the same primers, thermal profile, and PCR conditions specified above, with the exception of 3 mM MgCl2 and 4 µl SYBR Green (PE Applied Biosystems).

Immunocytochemistry

To visualize the distribution of immunoreactive chymase, a mouse antihuman mast cells chymase monoclonal MAB 1254 antibody was used (Chemicon International, Temecula, CA). The reaction was detected with Sigma Fast 3',3'-diaminobenzidine, 0.7 mg-tablets (DAB Tablets Set, Sigma). Serial 12-µm slices of mounted adrenal gland were fixed in acetone for 30 min at -20 C (17). Sections were washed in PBS (pH 7.4) and incubated with primary antibody to {alpha}-human-chymase (1:250) at 37 C for 40 min. After a 10 min wash, sections were incubated at 37 C for 40 min with a secondary peroxidase-conjugated rabbit antimouse IgG (1:40). They were rinsed again, and the reaction was developed for 5 min with 0.7 mg DAB tablets and stopped with water. Negative controls were carried out, in all cases, by similarly treating an adjacent section and omitting the primary antibody.

ET-1 assay

Adrenal-vein blood samples were collected from three patients for diagnostic purposes. Plasma (1 ml) was diluted with trifluoroacetic acid (TFA) and loaded on SePak columns (Waters Corp., Milford, MA). After washing, ETs were extracted with ethanol or acetonitrile acidified with acetic acid or TFA. The recovery ranged from 60–90% (18). The separation of ET-1[1–21] and ET-1[1–31] was performed by HPLC (Waters Corp. 600), using RP-18 reverse-phase column (Spherisorb ODS-2; Phase Separations Ltd, Deeside, UK) and a gradient H2O/acetonitrile with TFA for ionic suppression. A good separation of the two ET-1s was obtained, and the peptides were detected at 200- to 220-nm wavelength. A rough quantification of ET-1 was based on peak area measurement. The sensitivity of the assay was 1 pmol/ml, and intra- and interassay variation coefficients were 6.6% and 8.1%, respectively.

Steroid secretion

Dispersed cells were put in Medium 199 and Krebs-Ringer bicarbonate buffer with 0.2% glucose, containing 5 mg/ml human serum albumin. They were incubated (105 cells/ml) with increasing concentrations of ET-1[1–21] or ET-1[1–31] (from 10-12–10-7 M), and also with 10-8 M ET-1[1–31] in the presence of: 1) 10-4 or 10-5 M phosphoramidone; and 2) 10-7 M BQ-123 or BQ-788. The incubation was carried out at 37 C for 90 min in an atmosphere of 95% air-5% CO2. At the end of the experiments, the supernatants were obtained by centrifugation at 4 C and were stored at -80 C. Aldosterone and cortisol were extracted from the incubation media and purified by HPLC (19), and their concentrations were measured by RIA, using the following commercial kits: ALDO-CTK2 (IRE-Sorin, Vercelli, Italy; sensitivity 5 pg/ml) and cortisol-RIA (IRE-Sorin; sensitivity 30 pg/ml). Intra- and interassay variation coefficients were: aldosterone, 7.5% and 8.6%; and cortisol, 6.6% and 8.2%, respectively.

Adrenocortical cell DNA synthesis

Dispersed adrenocortical cells, obtained from three human adrenals, were suspended in Eagle’s MEM (Life Technologies, Paisley, UK), supplemented with 2% FBS, 100 U/ml penicillin, and 100 ng/L streptomycin, and plated in 35-mm tissue culture dishes at a density of 5 x 104 cells/dish. They were cultured at 37 C in a humidified atmosphere of 95% air-5% CO2 and employed after 24 h of culture (10). Cultures were incubated for 24 h as follows: 1) ET-1[1–21] or ET-1[1–31] (from 10-12–10-7 M); and 2) ET-1[1–21] or ET-1[1–31] (10-8 M) alone or in the presence of 10-6 M BQ-123 or BQ-788. During the last 12 h of incubation, BrdU was added to the culture medium at a final concentration of 20 mg/ml. Cultures were fixed in 4% paraformaldehyde for 30 min, and BrdU-positive (S-phase) cells were detected by immunocytochemistry (Cell Proliferation Kit; Amersham Pharmacia Biotech). Their number was evaluated by counting 3,000 cells per dish, and three dishes for each experimental point were employed.

Autoradiography

Segments of internal thoracic arteries, obtained from consenting patients undergoing coronary artery bypass graft surgery, and adrenal glands of six adult Wistar rats were immediately frozen at -30 C in isopentane and stored at -80 C. Frozen sections (10- to 15-µm thick) were cut in a cryostat (Leitz 1720 Digital, Leitz, Wetzlar, Germany) at -20 C and were processed as previously detailed (20). ET-1[1–21] binding sites in arteries were labeled in vitro by incubation for 120 min at 37 C with 10-9 M [125I]ET-1[1–21]. Nonspecific binding was determined by the addition of 10-7 M cold ET-1[1–21]. Selective [125I]ET-1[1–21] binding to ETA and ETB receptors was determined by the addition of 10-7 M BQ-788 and BQ-123, respectively, and the effect on it of 10-8 M ET-1[1–31] was studied. ET-![1–31] binding sites in adrenal glands were labeled, as detailed above, by incubating the sections with 5 x 10-9 M [125I]ET-1[1–31], and nonspecific binding was determined by the addition of 5 x 10-7 M cold ET-1[1–31]. Selective [125I]ET-1[1–31] to ETA and ETB receptors was assessed by the addition of 5 x 10-7 M BQ-123 or BQ-788, respectively. The reaction was stopped by washing the samples three times in 50 mM Tris/HCl buffer. After rinsing, the section were rapidly dried, fixed in paraformaldehyde vapors at 80 C for 2 h, and coated with Nuclear Track B2 nuclear emulsion (Eastman Kodak Co., Rochester, NY). Autoradiographs were exposed for 2 weeks at 4 C and then developed with undiluted Kodak D19 developer. They were stained with hematoxylin-eosin and photographed with a Leitz Laborlux microscope. Three unstained autoradiograms, taken from three arteries and three adrenal glands, were analyzed by computer-assisted densitometry with a camera-connected microscope and an IBM-compatible computer equipped with a software specifically written for this purpose (Studio Casti Imaging, Venice, Italy). For each autoradiogram, 10 areas of the vessel wall or of ZG and adrenal medulla (about 36,000 pixels) were analyzed. The density value of the lumen or the adrenal connective capsule was taken as the background value.

Statistics

Values were expressed as means ± SEM of three separate experiments. The statistical comparison of the results was done by ANOVA, followed by Duncan’s multiple-range test.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gene expression

RT-PCR, with primers specific for the human chymase gene, allowed detection of the mRNA of this gene in the human adrenal cortex as well as in the human myocardium (not shown) and internal thoracic artery (Fig. 1Go), two tissues used as positive control because they are known to possess a functional chymase activity. The result of an exemplary RT-PCR experiment showing the expression of the chymase gene in three different human adrenal cortexes, and in a specimen of internal thoracic artery taken as positive control, is shown in Fig. 1Go.



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  		Figure 1. Ethidium bromide-stained 2% agarose gel showing cDNA amplified with human chymase and GAPDH primers from mRNA of three different normal human adrenal cortexes (AG 1–3) and internal thoracic artery wall (ITA). Lanes 1 and 11 were loaded with 200 ng of a DNA size marker (Marker VIII; Boehringer Ingelheim GmbH, Mannheim, Germany). The amplified fragments were of the expected sizes: 552 bp for chymase, and 585 bp for GAPDH. No amplification of PCR mixture with water instead of RNA (lane 10), or without prior RT of the chymase mRNA is shown (lanes 12–15). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

 
Immunocytochemistry

Cryosections of five human adrenals incubated with an antibody specific to human chymase showed the expression of the enzyme in some cell clusters in the ZG and in the wall of capsular arterioles (Fig. 2Go).



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  		Figure 2. Cryosections of human adrenal cortex incubated with an antibody to human chymase, showing expression of the enzyme in islets of parenchymal cells in the ZG (A) and in the wall of a capsular arteriole (C). No immunostaining was seen in the section processed in an identical way with omission of the primary antibody (B). c, Gland connective capsule. Magnification, x20.

 
ET-1 assay

Both ET-1[1–21] and ET-1[1–31] were present in the adrenal vein blood, the concentration of the former being about 20-fold higher than that of the latter (58 ± 9 vs. 3.1 ± 0.5 pM).

Steroid hormone secretion

ET-1[1–21] concentration-dependently enhanced aldosterone and cortisol secretion from dispersed human ZG and ZF/R cells. Minimal and maximal effective concentrations were: aldosterone, 10-10 M and 10-8 M (1.5-fold and 3.4-fold rises); and cortisol, 10-11 M and 10-9 M (1.4-fold and 2.7-fold rises) (Fig. 3Go). ET-1[1–31] evoked a less intense secretory response that was significant only starting from a concentration of 10-8 M (1.4-fold and 1.8-fold rises for aldosterone and cortisol, respectively). ET-1[1–31] was significantly (P < 0.01) less potent than ET-1[1–21], EC50 being aldosterone: 2.2 ± 0.3 x 10-9 M vs. 3.5 ± 0.4 x 10-10 M; cortisol: 5.3 ± 0.8 x 10-9 M vs. 4.1 ± 0.7 x 10-10 M (Fig. 3Go). High concentrations of phosphoramidone (10-5 or 10-4 M) did not affect the secretory response of human adrenocortical cells to 10-8 M ET-1[1–31] (Fig. 4Go). BQ-123 and BQ-788 (10-7 M) partially blunted the secretagogue effect of 10-8 M ET-1[1–21] and, when administered together, annulled it (Fig. 5Go, upper panel). BQ-123 (10-7 M) suppressed the effect of 10-8 M ET-1[1–31] on both aldosterone and cortisol production, whereas BQ-788 was ineffective (Fig. 5Go, lower panel).



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  		Figure 3. Effects of ET-1[1–21] and ET-1[1–31] on aldosterone and cortisol secretion from dispersed human ZG and ZF/R cells. Data are the mean ± SEM of five independent experiments. +, P < 0.01 and *, P < 0.01 vs. the respective baseline (B) value.

 


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  		Figure 4. Lack of effect of phosphoramidone (PPR) on the secretory response of dispersed human ZG and ZF/R cells to ET-1[1–31]. Data are the mean ± SEM of three independent experiments. *, P < 0.01 vs. the respective baseline (B) value.

 


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  		Figure 5. Effects of BQ-123 and BQ-788 (10-7 M) on the secretory response of dispersed human ZG and ZF/R cells to ET-1[1–21] (upper panel) and ET-1[1–31] (lower panel). Data are the mean ± SEM of five independent experiments. +, P < 0.5 and *, P < 0.01 vs. the respective baseline (B) value; a, P < 0.05 and A, P < 0.01 vs. the respective control value

 
ZG cell DNA synthesis

Both ET-1[1–21] and ET-1[1–31] concentration-dependently raised the number of BrdU-positive cells in cultured human adrenocortical cells, indicating de novo DNA synthesis. Minimal and maximal effective concentrations were: ET-1[1–21], 10-10 M and 10-8 M (2.7-fold and 5.2-fold rises); and ET-1[1–31], 10-11 M and 10-8 M (4.9-fold and 10.0-fold rises), respectively. ET-1[1–31] was more potent than ET-1[1–21]: EC50, 4.2 ± 0.8 x 10-11 M vs. 3.5 ± 0.7 x 10-10 M; P < 0.01 (Fig. 6Go). The maximal stimulatory effect of both ET-1[1–21] and ET-1[1–31] was blocked by 10-6 M BQ-123 and unaffected by 10-6 M BQ-788 (Fig. 7Go).



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  		Figure 6. Effect of ET-1[1–21] and ET-1[1–31] on the proliferation rate of cultured human adrenocortical cells. Data are the mean ± SEM of three independent experiments. +, P < 0.05 and *, P < 0.01 vs. the respective baseline (B) value.

 


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  		Figure 7. Effect of BQ-123 and BQ-788 (10-6 M) on the proliferogenic response of cultured human adrenocortical cells to 10-8 M ET-1[1–21] or ET-1[1–31]. Data are the mean ± SEM of three independent experiments. *, P < 0.01 vs. the respective baseline (B) value; A, P < 0.01 vs. the respective control value.

 
Autoradiography

[125I]ET-1[1–21]-specific binding sites were present in the wall of human internal thoracic arteries, and were completely displaced by an excess of cold ET-1[1–21] (Fig. 8, A and BGo). Both BQ-123 and BQ-788 partially displaced binding, the effect of the former being much more intense than that of the latter (Fig. 8, C and DGo). ET-1[1–31] did not alter [125I]ET-1[1–21] binding to ETB receptors (labeled peptide plus BQ-123) (Fig. 8EGo) but abolished that to ETA receptors (labeled peptide plus BQ-788) (Fig. 8FGo). [125I]ET-1[1–31]-specific binding sites were present in both rat adrenal cortex and medulla (Fig. 9AGo). Binding was displaced by BQ-123 (Fig. 9BGo) and unaffected by BQ-788 (Fig. 9CGo). Quantitative densitometry confirmed these qualitative descriptions (Fig. 10Go).



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  		Figure 8. Autoradiographs of frozen sections of human internal thoracic-artery rings incubated with 10-9 M [125I]ET-1[1–21] alone (A) and in the presence of 10-7 M cold ET-1[1–21] (B), BQ-788 (C) or BQ-123 (D). 10-8 M ET-1[1–31] did not displace ET-1[1–21] binding to ETB receptors (labeled peptide plus BQ-123) (E), but eliminated binding to ETA receptors (labeled peptide plus BQ-788) (F). Magnification, x50.

 


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  		Figure 9. Autoradiographs of frozen sections of rat adrenal glands incubated with 5 x 10-9 M [125I]ET-1[1–31] alone (A) and in presence of 5 x 10-7 M BQ-123 (B) or BQ-788 (C). Only BQ-123 displaced ET-1[1–31] binding in both adrenal cortex and adrenal medulla (AM). Magnification, x70.

 


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  		Figure 10. Evaluation by quantitative densitometry of: 1) 10-9 M [125I]ET-1[1–21] binding in the human internal thoracic artery (total binding, TB), and of its displacement by cold ET-1[1–21] (10-7 M), and BQ-788 or BQ-123 (10-7 M) plus or without 10-8 M ET-1[1–31] (upper panel); and 2) 5 x 10-9 M [125I]ET-1[1–31] binding in the rat adrenal gland (TB), and of its displacement by cold ET-1[1–31] (5 x 10-7 M), and BQ-123 or BQ-788 (5 x 10-7 M) (lower panel) *, P < 0.01 vs. background (BG) value; a, P < 0.05 and A, P < 0.01 vs. TB value.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown that ET-1[1–21], at variance with other peptides that stimulate aldosterone secretion only at high concentrations (for review, see Ref.21), exerts such effect in vitro at concentrations that are consistent with a physiological role (22). In this study, we show that chymase is expressed in the human adrenal cortex. This enzyme has recently been implicated in the formation of a novel ET-1[1–31] peptide from bigET-1. Thus the demonstration and localization with gene expression and immunocytochemistry studies of chymase in the human adrenal cortex, as well as the detection of ET-1[1–31] in adrenal venous effluent, are the first novel findings of this study. They suggest that ET-1[1–31] can be synthesized locally in the human adrenal cortex and therefore raise the issue of the identification of a functional role of ET-1[1–31] in this gland.

Thus, we next examined the effects of this peptide on hormone secretion and DNA synthesis in human adrenals. Our results showed that ET-1[1–31] exerts a sizable stimulating action on steroid-hormone production by human adrenocortical cells, and confirmed previous experiments indicating that ET-1[1–21] has a similar effect by acting through both ETA and ETB receptor subtypes (22). Of much interest, ET-1[1–31] was far less potent than ET-1[1–21], its effect becoming significant only starting from a concentration of 10-8 M, i.e. 2–3 orders of magnitude higher than the minimal effective concentration of ET-1[1–21]. This might suggest that, to exert its secretagogue effect on human adrenocortical cells, ET-1[1–31] must be cleaved to ET-1[1–21] by ECE-1 (23), as found to occur in cultured bronchial smooth muscle cells (24). This possibility seems, however, unlikely, because high concentration of the ECE-1 inhibitor phosphoramidone did not abolish the secretagogue effect of ET-1[1–31]. We would like to suggest, instead, that the lower potency of ET-1[1–31], compared with ET-1[1–21], in eliciting a secretory response from human adrenocortical cells could be explained by assuming that ET-1[1–31] acts exclusively through ETA receptors. This latter contention is consistent with the following pieces of evidence: 1) the demonstration that maximal secretagogue effect of ET-1[1–21] on the human adrenal cortex requires the activation of both ETA and ETB receptors; 2) the fact that the adrenocortical secretagogue action of ET-1[1–31] was abolished by BQ-123 and unaffected by BQ-788; and 3) the lack of effect of ET-1[1–31] adrenocortical cell secretion in the rat (10), a species where the effect of ET-1[1–21] on steroid secretion is exclusively ETB-mediated (25). Finally, the contention that ET-1[1–31] is an exclusive agonist of ETA receptors is also supported by our autoradiographic data showing that: 1) ET-1[1–31] displaced [125I]ET-1[1–21] binding to ETA, but not ETB receptors, in human internal thoracic arteries; and 2) BQ-123, but not BQ-788, eliminated [125I]ET-1[1–31] binding in the rat adrenal gland.

Evidence has been provided that ETA receptors are mainly involved in the stimulation of the proliferative activity of rat ZG cells, through the activation of the tyrosine kinase- and PKC-dependent ERK1/2 cascade (10, 26). Hence, it seems reasonable to suggest that ET-1[1–31] could mainly act as adrenal growth promoter. This suggestion is supported by our present observation that ET-1[1–31] enhances adrenocortical-cell DNA synthesis more efficiently than ET-1[1–21], through the activation of ETA receptors as well as by the demonstration that ET-1[1–31] markedly stimulates proliferation of in vitro cultured human VSMCs through an ETA-mediated activation of PKC-dependent ERK1/2 cascade (9).

Collectively, our findings raise the possibility that, in human adrenals, the posttranslational alternative cleavage of bigET-1 by ECE-1 or chymase may lead, depending upon the local needs, to the production of either ET-1[1–21] or ET-1[1–31]; the former mainly stimulates adrenocortical steroidogenesis via both ETA and ETB receptors, the latter mainly exerts growth-promoting effects via ETA receptors. It remains to be established whether these mechanisms may be relevant for the onset of adrenocortical diseases characterized by both excess hormone secretion and cell proliferation, e.g. Conn’s adenomas, which express bigET-1 gene (27) and display a clear-cut secretory response to ET-1 (28).


   Acknowledgments
 


   Footnotes
 
Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; ECE, ET-converting enzyme; ET, endothelin; RT, reverse transcription; VSMC, vascular smooth muscle cells; ZF/R, zona fasciculata-reticularis; ZG, zona glomerulosa.

Received June 6, 2001.

Accepted September 21, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rubanyi GM, Polokoff MA 1994 Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev 46:325–415[Medline]
  2. Nakano A, Kishi F, Minami K, Wakabayashi H, Nakaya Y, Kido H 1997 Selective conversion of big endothelins to tracheal smooth muscle-constricting 31-amino acid-length endothelins by chymase from human mast cells. J Immunol 159:1987–1992[Abstract]
  3. Sanker S, Chandrasekharan UM, Wilk D, Glynias MJ, Karnik SS, Husain A 1997 Distinct multisite synergistic interactions determine substrate specificities of human chymase and rat chymase-1 for angiotensin II formation and degradation. J Biol Chem 272:2963–2968[Abstract/Free Full Text]
  4. Miyauchi T, Masaki T 1999 Pathophysiology of endothelin in the cardiovascular system. Annu Rev Physiol 61:391–416[CrossRef][Medline]
  5. Kishi F, Minami K, Okishima N, Murakami M, Mori S, Yano M, Niwa Y, Nakaya Y, Kido H 1998 Novel 31-amino acid length endothelins cause constriction of vascular smooth muscle. Biochem Biophys Res Commun 248: 387–390
  6. Yoshizumi M, Inui D, Okishima N, Houchi H, Tsuchiya K, Wakabayashi H, Kido H, Tamaki T 1998 Endothelin-1(1–31), a novel vasoactive peptide, increase [Ca2+]i in human coronary artery smooth muscle cells. Eur J Pharmacol 348:305–309[CrossRef][Medline]
  7. Inui D, Yoshizumi M, Okishima N, Houchi H, Tsuchiya K, Kido H, Tamaki T 1999 Mechanism of endothelin-1(1–31)-induced calcium signaling in human coronary artery smooth muscle cells. Am J Physiol 276:E1067–E1072
  8. Yoshizumi M, Inui D, Kirima K, Tsuchiya K, Houchi H, Azuma M, Yasuoka H, Kido H, Tamaki T 1999 Comparison of the effects of endothelin-1, -2 and -3 (1–31) on changes in [Ca2+]i in human coronary artery smooth muscle cells. Jpn J Pharmacol 81:298–304[CrossRef][Medline]
  9. Yoshizumi M, Kim S, Kagami S, Hamaguchi A, Tsuchiya K, Houchi H, Iwao H, Kido H, Tamaki T 1998 Effect of endothelin-1 (1–31) on extracellular signal-regulated kinase and proliferation of human coronary artery smooth muscle cells. Br J Pharmacol 125:1019–1027[CrossRef][Medline]
  10. Mazzocchi G, Rossi GP, Malendowicz LK, Champion HC, Nussdorfer GG 2000 Endothelin-1[1–31], acting as an ETA-receptor selective agonist, stimulates proliferation of cultured rat zona glomerulosa cells. FEBS Lett 487: 194–198
  11. Nussdorfer GG, Rossi GP, Malendowicz LK, Mazzocchi G 1999 The autocrine-paracrine endothelin system in the physiology and pathology of steroid secreting tissues. Pharmacol Rev 52:403–438
  12. Ohlstein EH, Elliott JD, Feuerstein GZ, Ruffolo Jr RR 1996 Endothelin receptors: receptor classification, novel receptor antagonists, and potential therapeutic targets. Med Res Rev 16:365–390[CrossRef][Medline]
  13. Loffler BM 2000 Endothelin converting enzyme inhibitors: current status and perspective. J Cardiovasc Pharmacol 35:S79–S82
  14. Andreis PG, Neri G, Prayer-Galetti T, Rossi GP, Gottardo G, Malendowicz LK, Nussdorfer GG 1997 Effects of adrenomedullin on the human adrenal glands: an in vitro study. J Clin Endocrinol Metab 82:1167–1170[Abstract/Free Full Text]
  15. Caughey GH, Zerweck EH, Vanderslice P 1991 Structure, chromosomal assignment, and deduced amino acid sequence of a human gene for mast cell chymase. J Biol Chem 266:12956–12963[Abstract/Free Full Text]
  16. Tso JY, Sun XH, Kao TH, Reece KS, Wu R 1985 Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res 13:2485–2502[Abstract/Free Full Text]
  17. Kleinjan A, Godthelp T, Blom HM, Fokkens WJ 1996 Fixation with Carnoy’s fluid reduces the number of chymase-positive mast-cells: not all chymase-positive mast cells are also positive for tryptase. Allergy 51:614–620[Medline]
  18. Savage P, Jeng AY 1993 Quantitation of plasma endothelins. Anal Biochem 213:434–436[CrossRef][Medline]
  19. Mazzocchi G, Rossi GP, Neri G, Malendowicz LK, Albertin G, Nussdorfer GG 1998 11ß-Hydroxysteroid dehydrogenase expression and activity in the human adrenal cortex. FASEB J 12:1533–1539[Abstract/Free Full Text]
  20. Rossi GP, Albertin G, Belloni AS, Zanin L, Biasolo MA, Prayer-Galetti T, Bader M, Nussdorfer GG, Palú G, Pessina AC 1994 Gene expression, localisation and characterisation of endothelin A and B receptors in the human adrenal cortex. J Clin Invest 94:1226–1234
  21. Nussdorfer GG 1996 Paracrine control of adrenal cortical function by medullary chromaffin cells. Pharmacol Rev 48:495–530[Medline]
  22. Rossi GP, Albertin G, Neri G, Andreis PG, Hofmann S, Pessina AC, Nussdorfer GG 1997 Endothelin-1 stimulates steroid secretion of human adrenocortical cells ex-vivo via both ETA and ETB receptor subtypes. J Clin Endocrinol Metab 82:3445–3449[Abstract/Free Full Text]
  23. Rossi GP, Albertin G, Zanin L, Franchin E, Sacchetto A, Cesari M, Palù G, Pessina AC 1995 Expression of the endothelin-converting enzyme gene (ECE-1) in human tissues. Biochem Biophys Res Commun 211:249–253[CrossRef][Medline]
  24. Hayasaki-Kajiwara Y, Naya N, Shimamura T, Iwasaki T, Nakajima M 1999 Endothelin generating pathway through endothelin1–31 in human cultured bronchial smooth muscle cells. Br J Pharmacol 127:1415–1421[CrossRef][Medline]
  25. Belloni AS, Rossi GP, Andreis PG, Neri G, Albertin G, Pessina AC, Nussdorfer GG 1996 Endothelin adrenocortical secretagogue effect is mediated by the B receptor in rats. Hypertension 27:1153–1159[Abstract/Free Full Text]
  26. Mazzocchi G, Rossi GP, Rebuffat P, Malendowicz LK, Markowska A, Nussdorfer GG 1997 Endothelins stimulate deoxyribonucleic acid synthesis and cell proliferation in rat adrenal zona glomerulosa, acting through an endothelin A receptor coupled with protein kinase C- and tyrosine kinase-dependent signaling pathways. Endocrinology 138:2333–2337[Abstract/Free Full Text]
  27. Rossi GP, Belloni AS, Albertin G, Zanin L, Biasolo MA, Nussdorfer GG, Palù G, Pessina AC 1995 Endothelin-1 and its receptors A and B in human aldosterone-producing adenomas. Hypertension 25:842–847[Abstract/Free Full Text]
  28. Rossi GP, Andreis PG, Neri G, Tortorella C, Pelizzo MR, Sacchetto A, Nussdorfer GG 2000 Endothelin-1 stimulates aldosterone synthesis in Conn’s adenomas via both A and B receptors coupled with the protein kinase C- and cyclooxygenase-dependent signaling pathways. J Invest Med 48:343–350[Medline]



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