1 and ET-3 Inhibit Estrogen and Camp Production by Rat Granulosa Cells in Vitro
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209 Endothelin (ET)-1 and ET-3 inhibit estrogen and cAMP production by rat granulosa cells in vitro A E Calogero, N Burrello and A M Ossino Division of Andrology, Department of Internal Medicine, University of Catania, 95123 Catania, Italy (Requests for offprints should be addressed to A E Calogero at Istituto di Medicina Interna e Specialita` Internistiche, Ospedale Garibaldi, Piazza S.M. di Gesu`, 95123 Catania, Italy) Abstract Endothelin (ET)-1 and ET-3, two peptides with a potent and a maximally stimulatory (3 mIU/ml) concentration of vasoconstrictive property, produce a variety of biological FSH. ET-1 and ET-3 dose-dependently suppressed basal effects in different tissues by acting through two different and FSH (1 mIU/ml)-stimulated cAMP production. ET-3 receptors, the ET-1 selective ETA receptor and the non- and SFX-S6c were significantly more potent than ET-1 in selective ETB receptor. An increasing body of literature suppressing estrogen production, suggesting that this effect suggests that ET-1 acts as a paracrine/autocrine regulator was not mediated by the ETA receptor. Indeed, BQ-123, of ovarian function. Indeed, ETB receptors have been a selective ETA receptor antagonist, did not influence identified in rat granulosa cells and ET-1 is a potent the inhibitory effects of ET-1 and ET-3 on basal and inhibitor of progesterone production. In contrast, incon- FSH-stimulated estrogen release. To determine a possible sistent data have been reported about the role of ET-1 on involvement of prostanoids, we evaluated the effects of estrogen production and the effects of ET-3 are not maximally effective concentrations of ET-1 and ET-3 on known. Therefore, the present study was undertaken to estrogen and cAMP production in the presence of in- evaluate the effects of ET-1 and ET-3 on estrogen and domethacin, a prostanoid synthesis inhibitor. This com- cAMP production, and the receptor type involved. Given pound did not have any effect on the suppressive effects of that prostanoids modulate ovarian steroidogenesis and that ETs on basal or FSH (1 mIU/ml)-stimulated estrogen or many actions of ETs are mediated by these compounds, we cAMP production. also evaluated whether the effects of ETs on estrogen and In conclusion, ET-1 and ET-3 were able to inhibit cAMP production might be prostanoid-mediated. ET-1, estrogen and cAMP production by rat granulosa cells, ET-3, and safarotoxin-S6c (SFX-S6c), a selective ETB indicating that the inhibitory effects of ETs on ovarian receptor agonist, inhibited basal estrogen production by steroidogenesis are not limited to progesterone biosyn- granulosa cells obtained from immature, estrogen-primed thesis. This effect does not appear to be mediated by female rats, in a concentration-dependent manner. All prostanoids or by the classical ETA and ETB receptors, at three peptides were also capable of inhibiting the produc- least under these experimental conditions. tion of estrogen stimulated by a half-maximal (1 mIU/ml) Journal of Endocrinology (1998) 157, 209–215 Introduction blocked by the peptide antagonist, BQ-123. Conversely, the ETB receptor binds ET-1 and ET-3 with equal affinity Endothelins (ETs), a family of 21-amino acid peptides, are and it is selectively activated by safarotoxin-S6c (SFX-S6c) present in three different isoforms coded by separate genes, (Bax & Saxena 1994). endothelin (ET)-1, ET-2, and ET-3 respectively (Inoue A number of studies have shown that ETs participate in et al. 1989). They were first isolated from endothelial the regulation of the endocrine system (Hirai et al. 1991, cells by virtue of their potent vasoconstrictor properties Masaki 1993, Calogero et al. 1994) and recent evidence (Yanagisawa et al. 1988). However, it soon became suggests that ETs may act as paracrine/autocrine regulators apparent that these peptides produce a variety of biological of ovarian endocrine function. Indeed, it has been shown effects (Masaki 1993) mediated by two distinct G-protein- that ETs are able to inhibit basal and gonadotropin- coupled receptors named ETA and ETB (Watanabe et al. stimulated progesterone production, cAMP accumulation, 1989). Although these receptors show about 63% amino and morphological transformation of granulosa cells in acid homology, they bind ET isopeptides and analogs with culture (Iwai et al. 1991, Flores et al. 1992, Tedeschi et al. different affinities. ETA has a more elevated affinity for 1992, 1994, Kamada et al. 1993). Although the effects ET-1 than for ET-3 and ETA-mediated cellular effects are of ETs on progesterone production are well known, Journal of Endocrinology (1998) 157, 209–215 1998 Society for Endocrinology Printed in Great Britain 0022–0795/98/0157–0209 $08.00/0 Downloaded from Bioscientifica.com at 09/23/2021 01:08:29PM via free access 210 A E CALOGERO and others · Endothelins and estrogen production inconsistent data have been reported on the role of ET-1 Bethesda, MD, USA), 35 µg/ml methyl-isobutylxanthine in estrogen production by granulosa cells (Kamada et al. (MIX; Sigma), and 1·25 µg/ml insulin (Eli Lilly Co., 1992, Tedeschi et al. 1994), and the effects of ET-3 are not Indianapolis, IN, USA). DES and MIX were added to known. Hence, this study was undertaken to evaluate the enhance the sensitivity of granulosa cells to FSH ( Jia & role of ET-1 and ET-3 on estrogen and cAMP production Hsueh 1986). At the end of the culture period, medium by granulosa cells obtained from immature, estrogen- samples were frozen at"20 )C until assayed for estrogen. primed rats, and to identify the ET receptor which medi- ates this effect. In consideration of the fact that prostanoids exert inhibitory effects on ovarian steroidogenesis Estrogen radioimmunoassay (Behrman & Caldwell 1986) and that a number of ET-1 The concentration of estrogen in the incubation medium actions are prostanoid-mediated (Anderson et al. 1995, was measured directly, without extraction, by radio- Howarth et al. 1995), we also evaluated whether indo- immunoassay, as previously reported (Calogero et al. methacin, a prostanoid synthesis inhibitor, modulates the 1996). Aliquots (200 µl) of medium, diluted 1:40–1:100, effects of ET-1 or ET-3 on estrogen or cAMP production. or standard solutions were incubated with 100 µl anti- serum (final dilution 1:10 000), and 100 µl [3H]estradiol Materials and Methods (E2) (about 9000 c.p.m.) at 4 )C for 18–20 h. Ice-cold charcoal-dextran was then added to achieve separation of Rat granulosa cell culture bound from free labeled hormone. Tubes were centrifuged at 1500 g at 4 )C for 11 min, the supernatants were Rat granulosa cells were cultured as previously reported collected and counted in a â-counter after the addition of ( Jia & Hsueh 1986, Calogero et al. 1996). Briefly, intact scintillation fluid. The anti-estrogen serum (RD/94/84; female immature (25 days old) Sprague-Dawley rats Analytical Antibodies, Segrate, MI, Italy) bound (Charles River, Calco, CO, Italy) were implanted sub- 3 40·7&2·5% (n=11) of [ H]E2. Non-specific binding cutaneously with a 10 mm Silastic brand capsule (Dow was 3·1&0·4%. The intra- and interassay coefficients of Corning, Midland, MI, USA) filled with diethylstilbestrol variation were 3·6&0·3% and 13·8% respectively. (DES; Sigma Chemical Co., St Louis, MO, USA). Four days after implantation, rats were sacrificed by cervical dislocation, and ovaries were removed for granulosa cell cAMP measurement collection. Ovaries were decapsulated, follicles were punc- tured with 27-gauge hypodermic needles, and granulosa At the end of the incubation, the medium was collected cells were carefully expressed into McCoy’s 5a medium for estrogen measurements and 0·5 ml ice-cold ethanol (Life Technologies, Paisley, Strathclyde, UK) to make a (90%) was added to each well. The plate was placed single cell suspension that was used for control and at"20 )C for 30 min and subsequently at 4 )C for 60– experimental cultures. An aliquot was diluted with trypan 90 min. Thereafter, each sample was collected and centri- blue stain, and viable cells were counted with a hemo- fuged in a microfuge, the supernatant collected, dried, cytometer. Cells were plated into 16 mm, 24-multiwell and stored at"20 )C until the day of the RIA. cAMP was plates (Corning, Milan, Italy) at a density of 50 000 viable measured by RIA using a commercially available kit (Amersham International plc, Amersham, Bucks, UK). cells/well/0·5 ml at 37 )C in a humidified, 95% air-5% cAMP antiserum bound 59·8&2·8% (n=2) of 125I-cAMP CO2 water-jacketed incubator in the presence of graded concentrations of ET-1 (Peninsula Laboratories Inc., (about 7000 c.p.m./100 µl). The detection limit (ED90)of Belmont, CA, USA), ET-3 (Peninsula), SFX-S6c (Sigma), the assay was 19·6 fmol/tube (49·0 fmol/well). The intra- BQ-123 (Peninsula), and/or indomethacin (Sigma) for six and interassay coefficients of variation were 1·3&0·3% days. BQ-123 or indomethacin were added simultaneously and 5·7% respectively. with ETs. Time-course studies showed that maximal estrogen production in response to increasing concen- Data analysis trations of follicle-stimulating hormone (FSH) (NIDDK hFSH B-1, AFP-8792B) was achieved after six days of Results, reported as means&... throughout the study, incubation (Calogero et al. 1996). Each treatment was are expressed as percentage of basal estrogen production. applied in triplicate within each experiment and each Statistical evaluation was performed by analysis of variance experiment was replicated at least three times. Incubation (ANOVA) followed by Duncan’s multiple range test. The was carried out using McCoy’s 5a medium supplemented effects of ET-1, ET-3, or SFX-S6c on basal and FSH- with 2 mM -glutamine (Life Technologies), 200 U/ml stimulated estrogen dose–response curves were analyzed penicillin, 200 µg/ml streptomycin sulfate, 0·5 µg/ml applying the four parameter logistic equation; a computer fungizone (Life Technologies), 10"6 M androstenedione program, ‘Allfit’, was employed for the computation (Sigma), 10"7 M DES (Sigma), 37·5 ng/ml human (DeLean et al.