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Endothelin-1 Stimulates Steroid Secretion of Human Adrenocortical Cells ex Vivo Via Both ET_A and ET_B Receptor Subtypes

Departments of Clinical and Experimental Medicine (G.P.R., G.A., S.H., A.C.P.) and Anatomy (G.N., P.G.A., G.G.N.), University of Padua, I-35121, Padua, Italy

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

	Abstract

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The role played by endothelins (ETs) and their receptor subtypes (ET_A and ET_B) in the regulation of steroid hormone secretion in human adrenal gland remains unclear. Therefore, we investigated the gene expression of ET-1 and its receptors in highly pure preparations of human adrenocortical cells and the effect of ET-1 on their secretory activity. Reverse transcription-PCR with primers specific for prepro-ET-1, ET-converting enzyme-1, ET_A, and ET_B complementary DNAs demonstrated the expression of all of these genes in human adrenocortical cells. ET-1 increased the secretion of aldosterone and cortisol by enhancing both earlier and late steps of their synthesis. The secretory response to ET-1 was partially (60%) inhibited by BQ-123 and BQ-788, which are selective antagonists of the ET_A and ET_B receptors, respectively. When added together, the two antagonists suppressed the secretagogue effect of ET-1. Collectively, these findings suggest that ET-1, acting via both ET_A and ET_B receptors, may exert an autocrine/paracrine regulation of the function of the human adrenal cortex.

	Introduction

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ENDOTHELIN-1 (ET-1) is the prototype of a family of 21-amino acid residue peptides that exerts multiple biological effects, including very potent vasoconstriction, inhibition of renin release by kidney juxtaglomerular cells, and stimulation of catecholamine, vasopressin, and aldosterone secretion (for review, see Refs. 1–3). The latter effect was observed both in vivo and in vitro and is potentially important in conditions where enhanced ET-1 and aldosterone secretions coexist, such as severe and/or malignant hypertension, congestive heart failure, and hypoxia (for review, see Refs. 2 and 4).

We recently demonstrated expression of the genes for prepro-ET-1 and its receptor subtypes, ET_A and ET_B, in homogenates of the normal human adrenal cortex and aldosterone-producing tumors, and autoradiography with specific antagonists of ET_A and ET_B receptors confirmed that both ET receptor subtypes are translated into functional proteins in these tissues (5, 6). However, these studies did not allow us to conclusively ascertain whether specific messenger ribonucleic acids (mRNAs) and ET receptors are contained in the parenchymal steroid-secreting cells or in the stromal (connective and vascular) components of the gland. The exclusive stromal location of ET receptors appears to be ruled out by the occasional demonstration that ET-1 elicits a marked secretory response in dispersed human adrenocortical cells (7). Unfortunately, the lack of specific ET receptor antagonists prevented these investigators to settle which subtype of receptor mediates this effect of ET-1.

It, therefore, seemed worthwhile to perform a study aimed at investigating whether 1) dispersed human adrenocortical cells express the genes for prepro-ET-1, human ET-1 converting enzyme-1 (hECE-1), and ET_A and ET_B receptors, and 2) the receptor subtype involved in their secretory response to ET-1.

	Materials and Methods

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Reagents

Moloney murine leukemia virus reverse transcriptase (Gene Amp RNA PCR core kit) and Taq polymerase (AmpliTaq) were purchased from Perkin-Elmer/Cetus (Norwalk, CT). ET-1 was obtained from either Peninsula Laboratories (Merseyside, UK) or Neosystem Laboratories (Strasbourg, France). BQ-123, a selective ET_A antagonist, and BQ-788, a selective ET_B antagonist (8), were purchased from Neosystem Laboratories. Medium 199 was obtained from Difco (Detroit, MI), ACTH, angiotensin II (ANG-II), human serum albumin, and other laboratory reagents were obtained from Sigma Chemical Co. (St. Louis, MO); cyanoketone (WIN 24540) was purchased from Sterling-Winthrop (Guilford, UK); and RIA kits for aldosterone and cortisol were obtained from IRE-Sorin (Vercelli, Italy).

Dispersed human adrenocortical cells

Dispersed cells were obtained from adrenal glands removed from six consenting patients undergoing unilateral nephrectomy with ipsilateral adrenalectomy for renal cancer. Starting from 2 weeks before surgery, patients were kept on a normal diet; only patients not requiring medications able to alter adrenal function were recruited. Specimens were collected immediately after excision in the operating room, placed in Krebs-Ringer bicarbonate buffer with 0.2% glucose at 4 C, and immediately carried to the cell biology laboratory, where cell isolation was immediately undertaken by collagenase digestion and mechanical disaggregation as previously reported (9). The contamination of our adrenocortical cell preparations by stromal elements, as evaluated by phase microscopy, was virtually absent, and the viability of isolated cells, as checked by the trypan blue exclusion test, was higher than 92%. Adjacent sections of the adrenal gland underwent pathological evaluation and were found to be histologically normal. The study protocol followed the local ethical committee guidelines for human studies.

RNA extraction and reverse transcription-PCR (RT-PCR)

Freshly isolated cells were used for RNA extraction with the guanidium isothiocyanate method. After isolation, total RNA was checked for integrity by 1.5% agarose gel electrophoresis and quantified by measurement of UV absorbance at 260 nm.

For use in the PCR, total RNA was reversely transcribed to complementary DNA (cDNA) with random hexamers (2.5 µmol/L) and 50 U cloned Moloney murine leukemia virus reverse transcriptase, as previously reported in detail (5). Amplification of the prepro-ET-1, ET_A, ET_B, and hECE-1 cDNAs was carried out with the primers and the thermal profiles previously reported (5, 10), using a Delphi 100 Thermal Cycler (Oracle Biosystem, MJ Research, Waterston, MA). Specificity of the amplification products for the gene of interest was confirmed by hybridization with the following cDNA-specific probes: hECE-1, 5'-TTG GAC TTT GAG ACG GCA CTG GC-3'; ET_A receptor, 5'-CCT CAA CCT CTG CGC TCT TAG TGT-3'; and ET_B receptor, 5'-TCC TGC CTT GTG TTC GTG CTG GGG-3'. As a positive control, amplification of a 838-bp fragment of the human ß-actin gene was carried out in parallel (5). As false positive results of RT-PCR for the ß-actin gene, due to amplification of retropseudogenes, have been previously reported (11), in parallel experiments ET_A and ET_B receptor PCRs were carried out with no prior reverse transcription to further rule out the possibility of amplifying genomic DNA.

Steroid secretion

Dispersed cells were put in medium 199 and Krebs-Ringer bicarbonate buffer with 2% glucose containing 5 mg/mL human serum albumin and incubated (3 x 10⁵ cells/mL, in replicates of five each) as follows: 1) ACTH, ANG-II, or ET-1 (all 10^-9 mol/L) in the presence or absence of 10 µmol/L cyanoketone, and 2) ET-1 (10^-9 mol/L) or ANG-II (10^-9 mol/L) in the presence or absence of 10^-7 mol/L BQ-123 and/or BQ-788. The concentrations of ACTH, ANG-II, ET-1, and ET receptor antagonists were those determined to be maximally effective in vitro (12, 13), and that of cyanoketone was previously found to completely prevent further metabolism of pregnenolone (14). Incubations were carried out in a shaking bath at 37 C for 90 min in an atmosphere of 95% air-5% CO₂. The medium was collected and kept frozen at -80 C until hormonal assays.

In the first incubation experiment, the concentrations of pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, 18-hydroxycorticosterone, aldosterone, 11-deoxycortisol, cortisol, and cortisone were measured by high pressure liquid chromatography (HPLC), as previously described (9). Pregnenolone was detected by UV absorbance at 290 nm (14), and the other steroids were detected at 240 nm wavelength and identified by comparison of their retention times with those of the standards. A good separation between steroid hormones assayed was obtained (9, 14), and the final recovery of steroid was 80–85%. Quantification of steroid hormones was based on peak area measurement; the sensitivity of our assay system was 1 pmol/mL, and the response of the detector was satisfactorily linear over the range of 1–1000 pmol and was directly proportional to the mass of steroid hormone injected. Intra- and interassay variations were 5.1% and 7.2%, respectively.

In the second incubation experiment, aldosterone and cortisol concentrations were measured by RIA, after extraction and HPLC purification, with the ALDO-CTK2 (sensitivity, 5 pg/mL; cross-reactivity: aldosterone, 100%; 17-iso-aldosterone and other steroids, <0.1%; intra- and interassay variations, 7.5% and 8.9%) and cortisol RIA (sensitivity, 30 pg/mL; cross-reactivity: cortisol, 100%; 11-deoxycortisol, 4.8%; corticosterone, 3%; progesterone, 0.5%; 11-deoxycorticosterone, 0.02%; other steroids, <0.01%; intra- and interassay variations, 6.2% and 7.8%) kits.

Data obtained from each adrenal gland were averaged and expressed as the mean ± SD or SEM of six separate experiments (six adrenals from six patients). The statistical comparison of results was performed using ANOVA, followed by the multiple range test of Duncan.

	Results

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Gene expression

RT-PCR analysis of RNA from dispersed human adrenocortical cells showed prepro-ET-1, ET_A, ET_B, and hECE-1 mRNAs in all samples examined. The specificity of the amplification products obtained was confirmed by 1) hybridization with cDNA-specific probes, 2) size identity, and 3) lack of amplification of each cDNA when diethyl pyrocarbonate water was used instead of mRNA as template for the RT-PCR (Fig. 1).

View larger version (27K): [in this window] [in a new window]   Figure 1. Ethidium bromide-stained 1.5% agarose gel showing cDNA amplified with human prepro-ET-1-specific, hECE-1-specific (A), ETA- and ETB-specific (B) primers from RNA of different, freshly isolated human adrenocortical cells. C shows the different amplification products of prepro-ET-1, hECE-1, ETA, and ETB and also of aldosterone synthase from cells of the same individual. Lane 1 was loaded with 200 ng of a size marker (Boehringer Mannheim, marker VIII). The amplified fragments were of the expected sizes, which was 442 bp for prepro-ET-1, 567 bp for hECE-1 669 bp for ETA, and 760 bp for ETB. No amplification of PCR mixture with no cDNA template is also shown as a negative control. Amplification of an 838-bp fragment of ß-actin is also shown as a positive control.

The HPLC assay (Table 1) showed that dispersed human adrenocortical cells secrete significant basal amounts of both glucocorticoid and mineralocorticoid hormones. ACTH and ANG-II (1 nmol/L) stimulated both early and late steps of steroid synthesis, but the effect of the latter agonist was significantly less intense (the rises in pregnenolone, cortisol, and aldosterone production were 8.9- vs. 3.2-fold, 8.1- vs. 2.6-fold, and 10.7- vs. 4.3-fold, respectively). ET-1 (1 nmol/L) also enhanced both early and late steps of steroid synthesis; its effectiveness was of about the same order of magnitude of that of ANG-II (the rises in pregnenolone, cortisol, and aldosterone were 2.8-, 2.9-, and 5.0-fold, respectively).

View larger version (23K): [in this window] [in a new window]   Figure 2. Effects of BQ-123 (10-7 mol/L) and/or BQ-788 (10-7 mol/L) on basal (B) and ET-1 (10-9 mol/L)- or ANG-II (10-9 mol/L)-stimulated aldosterone and cortisol secretion by dispersed human adrenocortical cells. Data are the mean ± SEM (n = 6). a, P < 0.05; A, P < 0.01 (vs. the respective basal value). *, P < 0.01 (vs. the respective control value).

	Discussion

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These results suggest that human adrenocortical cells are endowed with a functional ET-1 biosynthetic pathway, a finding in agreement with the immunocytochemical demonstration of the presence of ET-1-positive steroid-secreting cells in normal human adrenal cortex (15). They also rule out the recently reported exclusive extraparenchymal location of ET-1 in human adrenals (16). Moreover, our findings clearly demonstrate that the ET_A and ET_B receptor subtypes evidenced by autoradiography in the human adrenal cortex (5, 16) are at least in part located on steroid-secreting cells.

Our dispersed cell preparations secrete sizable basal amounts of glucocorticoids and mineralocorticoids, and, as expected, respond not only to ACTH, but also to ANG-II. In fact, human zona fasciculata-reticularis cells, at variance with those of rodents, possess the AT1 subtype of ANG-II receptors functionally coupled with cortisol secretion (17). Our functional experiments confirm the earlier observation (7) that ET-1 (1 nmol/L) exerts a clear-cut direct glucocorticoid and mineralocorticoid secretagogue effect on dispersed human adrenocortical cells, which is about as potent as that elicited by an equimolar concentration of ANG-II. Moreover, they show that ET-1 stimulates not only the late steps of aldosterone and cortisol synthesis, but also the early rate-limiting one, i.e. the conversion of cholesterol to pregnenolone. This last observation appears to be in keeping with the recent demonstration that ET-1 enhances both the uptake and the utilization of cholesterol in steroid synthesis by rat adrenal cortex (18).

Recent data from our laboratories unequivocally indicated that dispersed rat adrenocortical cell express only the ET_B receptor gene, and that the secretory responses to ETs are exclusively mediated via this receptor subtype (13, 19). This latter finding accords well with the in vitro experiments carried out on dispersed calf zona glomerulosa cells (20, 21). In contrast, the present results clearly demonstrate that both ET_A and ET_B receptors mediate the secretory response to ET-1 of human adrenocortical cells. In fact, both the ET_A antagonist BQ-123 and the ET_B antagonist BQ-788 partially inhibit the secretagogue effect of ET-1, and when added together they abolish it. The two antagonists do not alter either basal or ANG-II-stimulated secretion of aldosterone and cortisol, thereby making unlikely the possibility of an aspecific toxic effect on the steroidogenic machinery or an interference with the intracellular mechanisms transducing the ANG-II secretagogue signal.

In conclusion, our present results provide evidence that human adrenocortical cells are able to synthesize ET-1 and possess ET_A and ET_B receptors functionally coupled with steroid hormone secretion, whose activation, however, is not required for ANG-II to exert its stimulatory effect. Collectively, these findings suggest an autocrine/paracrine action of ET-1 in the regulation of human adrenocortical functions in vivo.

Received April 8, 1997.

Revised June 19, 1997.

Accepted July 2, 1997.

	References

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