Endothelins Enhance Prostaglandin (PGE2 and PGF2 ) Biosynthesis ...

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cells exposed to graded doses of ET-1 and ET-3; PGF2α and PGE2 were ... ET-3. Experiments with two endothelin receptor antagonists (the. BQ485 and BQ788 ...
0021-972X/01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2001 by The Endocrine Society

Vol. 86, No. 2 Printed in U.S.A.

Endothelins Enhance Prostaglandin (PGE2 and PGF2␣) Biosynthesis and Release by Human Luteal Cells: Evidence of a New Paracrine/Autocrine Regulation of Luteal Function FIORELLA MICELI, FRANCESCA MINICI, MARINA GARCIA PARDO, PIERLUIGI NAVARRA, CATERINA PROTO, SALVATORE MANCUSO, ANTONIO LANZONE, AND ROSANNA APA Departments of Obstetrics and Gynecology (Fi.M., Fr.M., M.G.P., S.M., R.A.) and Pharmacology (P.N.), Universita` Cattolica del Sacro Cuore, 00168 Rome, Italy; and OASI Institute for Research (C.P., A.L.), 94018 Troina, Italy ABSTRACT We have previously shown that endothelin-1 (ET-1) is normally found in human luteal cells, where it is able to significantly inhibit both basal and hCG-induced progesterone production. To further expand our comprehension of the possible roles of endothelins (ETs) in luteal physiology, in this study we used primary cultures of luteal cells exposed to graded doses of ET-1 and ET-3; PGF2␣ and PGE2 were assayed in the culture medium to investigate whether ETs also influence cyclooxygenase activity in these cells. We found that both ETs are able to significantly stimulate PGF2␣ and PGE2 release in a doseand time-dependent manner. ET-1 was always more effective than

ET-3. Experiments with two endothelin receptor antagonists (the BQ485 and BQ788 compounds, which block the ET-A and ET-B receptors, respectively) showed that the two endothelins induce PG production through different receptors and signaling pathways. In conclusion, here we demonstrate the ability of ETs to influence PG synthesis and release from human luteal cells. As PGs are deeply involved in corpus luteum activity, and ETs were also able to influence progesterone production, the present new data suggest an interesting interplay among progesterone, PGs, and ETs in the control of corpus luteum physiology. (J Clin Endocrinol Metab 86: 811– 817, 2001)

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significantly inhibit basal and hCG-stimulated P production (8, 9). Two ET receptor subtypes, ET-A and ET-B, have been identified in a variety of tissues. ET-1 has a higher selectivity for ET-A; its binding affinity is almost 100 times higher than that of ET-3 (10), whereas ET-B is nonselective and is bound by all ETs with similar affinities (11, 12). ET-1 binds the ET-A and ET-B receptors with equal affinities, although the majority of its effects are mediated by ET-A receptors. The latter are coupled, via G protein, to the activation of phospholipase C in all tissues investigated (7, 13, 14). Conversely, in cultured porcine granulosa cells ETs attenuate gonadotropinstimulated P secretion by inhibiting adenylyl cyclase, and this effect is mediated by the ET-B subtype, indicating that the latter is coupled to activation of the adenylyl cyclasecAMP pathway (15). It is well established that the regression of CL is essential for the normal menstrual cycle because it allows the development of a new ovulatory follicle, whereas prevention of luteolysis is necessary for the maintenance of pregnancy. In CL physiology, a key role is played by both PGF2␣ and PGE2. The latter seems to exert a luteotropic action; in animals it stimulates luteal P production (16), and in humans a positive correlation between P and PGE2 has been demonstrated throughout the menstrual cycle (17). PGE2 was also shown to stimulate P synthesis (18, 19) and activate the cAMP pathway (18, 20) in human CL in vitro. Conversely, PGF2␣ seems to be involved in CL regression; a negative correlation has

HE PROCESS OF initiation and maintenance of corpus luteum (CL) function requires fine-tune regulation of hormonal signaling by nonsteroidal intraovarian factors acting in a autocrine or paracrine manner. Compelling evidence indicates that endothelins (ETs) may play a role as local modulators of ovarian function. A potent vasoconstrictor factor originally isolated from porcine endothelial cells (1), ET-1 is a 21-amino acid peptide and a member of a family of structurally homologous peptides that also includes ET-2 and ET-3 (2). Although ETs are the products of three different genes, they share extensive sequence homology and a common structural design (3). Recently, ETs and their receptors have been localized in the human endometrium, where they are differentially expressed throughout the menstrual cycle (4). In the human ovary, elevated concentrations of ET-1 were found in follicular fluid (5, 6), with higher levels in gonadotropin-induced than in spontaneous cycles. ET-1 messenger ribonucleic acid (mRNA) has been detected in human granulosa cells, where the peptide was shown to inhibit basal, FSH-stimulated, and LH-stimulated progesterone (P) synthesis (6, 7). We recently observed that human luteal cells also express ET-1 mRNA, and that the peptide is able to Received April 27, 2000. Revision received August 2, 2000. Rerevision received September 30, 2000. Accepted October 13, 2000. Address all correspondence and requests for reprints to: Rosanna Apa, M.D., Department of Obstetrics and Gynecology, Universita` Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, Italy. E-mail: [email protected].

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been demonstrated between the concentrations of P and PGF2␣ throughout the human menstrual cycle (21), and the intraluteal injection of PGF2␣ caused both an immediate fall in serum P and a shortening of the luteal phase (22). The present study was designed to continue our investigation about the role played by ETs in CL physiology; in particular, we sought to determine whether these peptides are able to influence PGs synthesis in human luteal cells in vitro. Materials and Methods Chemicals Synthetic ET-1 and ET-3 were obtained from Roche (Mannheim, Germany); BQ485 and BQ788 were purchased from NovaBiochem (La Jolla, CA). hCG and phorbol 12-myristate 13-acetate (PMA) were purchased from Serono (Rome, Italy). The following chemicals were purchased from Sigma (St. Louis, MO): collagenase type IV, antibiotics, glutamine, HEPES and cAMP. Ham’s F-12 medium was obtained from Flow Laboratories (Milan, Italy), and FCS was purchased from Biological Industries (Kibbutz Beit HaEmek, Israel).

Luteal cell culture preparation and experimental procedure CL were obtained at the time of hysterectomy performed for nonendocrine gynecological diseases (leiomyomatosis) in the midluteal phase of the menstrual cycle (days 5– 6 from ovulation). Patients were between 30 – 43 yr of age. All of them had a history of regular menstrual cycles. A total of 13 experiments were performed. Informed consent was obtained from each patient, and the study was approved by the internal review board. The age of the CL was determined as follows. All patients were monitored until ovulation by daily measurement of basal body temperature (BBT) and ultrasound examination of follicular growth. When the maximal follicular diameter had reached 18 mm, daily determination of plasma P values was made. The time of ovulation (day 0) was detected by the biphasic pattern of BBT, by the typical ultrasound disappearance of the dominant follicle or the ultrasound detection of CL, and by the rise in plasma P concentrations. At the time of surgery, plasma samples were collected immediately before anesthesia to determine plasma P concentrations. The luteal tissue removed was immediately freed from blood vessels and ovarian stroma under a dissecting microscope, dissected, and minced. Human CL cultures were performed as previously described (23) with some modification. The luteal tissue was placed in 10 mL prewarmed Ham’s F-12 HEPES medium containing type IV collagenase (200 U/mL), then incubated at 37 C in a shaking water bath for 45 min. The medium containing the cell suspension was filtered through a 40-␮m nylon mesh, and the cells obtained were centrifuged and resuspended twice in fresh medium. This procedure was repeated once with the remaining undigested tissue to obtain highly purified luteal cells. Cells were counted in a hemocytometer, and viability was determined by the trypan blue exclusion test. The cells were diluted to a final concentration of 250,000 live cells/mL medium supplemented with 2 mmol/L l-glutamine, 100 IU penicillin, 100 mg/mL streptomycin, and 10% FCS and cultured in 48-multiwell plates for 24 h in 5% CO2 and 95% air at 37 C. After this time the cells were attached to the wells; the medium was then removed and replaced with fresh serum-free medium alone (controls) or containing ET-1 (10⫺9–10⫺6 mol/ L), ET-3 (10⫺9–10⫺6 mol/L), BQ 485 (10⫺9–10⫺6 mol/L) alone or combined with ET-1 (10⫺7 mol/L), and BQ 788 (10⫺9–10⫺6 mol/L) alone or combined with ET-3 (10⫺7 mol/L). In another group of experiments, we stimulated the cells with cAMP (1 mmol/L) and PMA (10⫺7 mol/L) either alone or combined with ET-1 or ET-3 (10⫺7 mol/L). At the end of experiments the cells were stained for lipids with oil red O (14) and counted. More than 90% of the luteal cells stained positively for lipids. The remainder did not stain for lipids, and there were occasional vascular cells, including erythrocytes and leukocytes. The medium was harvested after 12, 24, and 48 h of culture and stored at ⫺20 C until assayed for PG immunoreactivity.

PG assays RIAs for PGE2 and PGF2␣ adopted in this study were first characterized for measurement of the prostanoids in human urine (24) and later were used successfully to measure PGs produced and released by several cell types in vitro, including cells from human ovaries (20). Briefly, for each assay, incubation mixtures of 1.5 mL were prepared in disposable plastic tubes in which 50 ␮L incubation medium were diluted to 250 ␮L with 0.025 mol/L phosphate buffer (pH 7.5). Tritiated PGE2 or PGF2␣ (2,500 –3,500 cpm) and appropriately diluted antisera were added together to a final volume of 1.5 mL. The antisera (provided by Prof. G. Ciabattoni) were employed at a final dilution of 1:120,000 or 1:150,000 (for PGE2 or PGF2␣, respectively). A duplicate standard curve ranging from 2–100 pg/tube was run for each assay. All tubes were incubated for 24 h at 4 C. Separation of antibody-bound PGs was obtained with 2.5 mg charcoal (Norit-A), which absorbs 95–98% of free PGs; a charcoal suspension (2.5 mg/50 ␮L) in 0.025 mol/L phosphate buffer, pH 7.5, was added to each tube after the addition of 100 ␮L 5% BSA. The tubes were briefly shaken and then centrifuged for 10 min at 4 C. Supernatants were decanted into 10 mL scintillation liquid. Radioactivity was measured by liquid scintillation counting. The detection limit of the assay was 2 pg/tube in all cases. The inter- and intraassay variability coefficients were 2.7% and 2.9% for PGE2, and 3.2% and 2.8% for PGF2␣, respectively. [3H]PGE2 and [3H]PGF2␣ were obtained from NEN Life Science Products (Milan, Italy).

Data analysis Data were first analyzed by Kolmorogov-Smirnov test to assess differences in the general shapes of distribution. Normally distributed data were then analyzed by one-way ANOVA with Bonferroni correction to perform pairwise comparisons between group means.

Results

Both PGF2␣ and PGE2 are produced and released in sizable amounts from human luteal cells. After release, PGs do not appear to be further taken up or metabolized by cultured cells, as they tend to accumulate within the incubation medium in a time-related fashion (Fig. 2). As PGs are not stored in these cells, the fraction released in the medium can be taken as a reliable index of total PG biosynthesis. Effects of ET-1 and ET-3 on PGF2␣ and PGE2 synthesis by human luteal cells

Luteal cells were cultured for 24 h with graded doses of ET-1 and ET-3 (10⫺9–10⫺6 mol/L). As shown in Fig. 1A, both ETs were able to increase PGF2␣ release in a dose-dependent manner. The effect was statistically significant (P ⬍ 0.05) for ET-1 from 10⫺8 mol/L onward, whereas for ET-3 it was significant from 10⫺7 mol/L onward. In a similar way, both ETs positively affected PGE2 production (Fig. 1B) with the same potencies as those observed for PGF2␣. In fact, in this case also ET-1 significantly stimulated PGE2 release from 10⫺8 mol/L (P ⬍ 0.05) onward, whereas ET-3 was effective from 10⫺7 mol/L (P ⬍ 0.001). A plateau was reached by both ETs at a concentration of 10⫺7 mol/L. The next step was to investigate whether the effect of ETs was time dependent. To this purpose, luteal cells were incubated with 10⫺7 mol/L ET-1 and ET-3 for 12, 24, and 48 h. Figure 2 (left panel) shows how the highest PGF2␣ levels were induced by ET-1 after 24 h of culture (P ⬍ 0.001 vs. controls) even though a significant effect was already present after 12 h (P ⬍ 0.05). After 48 h of incubation, PGF2␣ concentrations fell to values similar to those observed in the control group. The effect of ET-3 was statistically significant only after 24 h of

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FIG. 1. A, Dose-dependent effects of ET-1 and ET-3 on PGF2␣ levels measured in incubation medium of human luteal cells. The latter were cultured for 24 h with medium alone (CTR) or with increasing concentrations of ET-1 and ET-3 (10⫺9–10⫺6 mol/L). Data represent the mean ⫾ SEM of eight experiments. Significance vs. CTR: *, P ⬍ 0.05; ***, P ⬍ 0.001. B, Dose-dependent effects of ET-1 and ET-3 on PGE2 levels measured in incubation medium of human luteal cells. The latter were cultured for 24 h with medium alone (CTR) or with increasing concentrations of ET-1 and ET-3 (10⫺9–10⫺6 mol/L). Data represent the mean ⫾ SEM of eight experiments. Significance vs. CTR: *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001.

culture (P ⬍ 0.05), whereas no effect was seen at 12 and 48 h. ET-1 was overall more effective than ET-3 in stimulating PGF2␣ release, as it was already effective after 12 h of culture, and at 24 h its effect was stronger compared with that of ET-3 (ET-1 vs. controls, P ⬍ 0.001; ET-3 vs. controls, P ⬍ 0.05). Figure 2 (right panel) shows the time-dependent effects of both ETs on PGE2 release. ET-1 exerted a time-dependent effect that was already significant after 12 h (P ⬍ 0.05) and further increased after 24 (P ⬍ 0.001) and 48 h (P ⬍ 0.01 vs. controls). ET-3 also significantly stimulated PGE2 at 12 (P ⬍ 0.05 vs. controls) and 24 h (P ⬍ 0.001), whereas at 48 h PG

levels were similar to those observed in the control group. Only at 48 h was ET-1 more effective than ET-3. Receptor subtypes mediating the effects of ETs on PGs release

To determine the subtype(s) of receptor mediating the effects of ETs on PG release, luteal cells were preincubated for 30 min with BQ 485 (10⫺9–10⫺6 mol/L) or BQ 788 (10⫺9– 10⫺6 mol/L). The former is a potent and selective ET-A receptor antagonist, whereas BQ 788 selectively blocks ET-B

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FIG. 2. Time-dependent effects of ET-1 and ET-3 on PGF2␣ (left panel) and PGE2 (right panel) levels measured in incubation medium of human luteal cells. The latter were cultured for 12, 24, and 48 h with medium alone (CTR) or in the presence of ET-1 or ET-3 (10⫺7 mol/L). Data represent the mean ⫾ SEM of eight experiments for the 24-h period and of six experiments for the 12- and 48-h periods. Significance vs. CTR: *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001.

FIG. 3. Dose-dependent reversal of the ET-1-stimulated PGF2␣ (left panel) and PGE2 (right panel) levels by BQ485, an ET-A receptor antagonist. Luteal cells were cultured for 24 h with medium alone (CTR), ET-1 (10⫺7 mol/L), or BQ485 (10⫺6 mol/L) or were preincubated for 30 min with increasing concentrations of BQ485 (10⫺9–10⫺6 mol/L) and then treated with ET-1 (10⫺7 mol/L). Data represent the mean ⫾ SEM of six experiments. Significance vs. CTR: ***, P ⬍ 0.001. Significance vs. ET-1: E, P ⬍ 0.05; EE, P ⬍ 0.01; EEE, P ⬍ 0.001.

receptors. Cells were then treated for 24 h with ET-1 (the groups receiving BQ 485) and ET-3 (the groups receiving BQ 788), both at a concentration of 10⫺7 mol/L. As shown in Fig. 3, in the presence of BQ 485 the stimulatory effect of ET-1 on PGF2␣ (left panel) and PGE2 release (right panel) was dose dependently reduced; at its highest concentration, BQ 485 completely abolished stimulation by ET-1. A remarkably similar effect was observed when the cells were incubated with ET-3 and the ET-B receptor antagonist BQ788 (Fig. 4). Also in this case, we observed a graded decrease in ET-3induced PG release that paralleled the increase in BQ788

concentrations. At the highest concentration of BQ 788, the effect of ET-3 was completely abolished. Neither receptor antagonist had an intrinsic effect on PGs release when given alone. Signal transduction pathways

In an attempt to clarify the pathway(s) through which ETs exert their effects on PGs, luteal cells were incubated for 24 h with PMA (100 ng/mL) or cAMP (1 mmol/L) alone or combined with ET-1 and ET-3 (10⫺7 mol/L). It is interesting to

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notice that the results obtained for PGF2␣ (Fig. 5A) and PGE2 (Fig. 5B) were superimposable. We found that both PMA and cAMP, when given alone, produced a much higher increase in PGs release compared with control and ET-treated groups (cAMP, P ⬍ 0.001 vs. control and ET groups; PMA, P ⬍ 0.001 vs. control and ET groups). When they were used in association with the ETs, we found that only ET-1 was able to further increase the positive effect of cAMP on both PGs (P ⬍

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0.05 vs. cAMP alone). In all other cases, the ETs were unable to further enhance cAMP- or PMA-stimulated PGs release. Discussion

In addition to gonadotropins, new factors have recently thought to be involved in CL physiology, as classic endocrine theories cannot fully explain all aspects of CL function. In-

FIG. 4. Dose-dependent reversal of the ET-3-stimulated PGF2␣ (left panel) and PGE2 levels (right panel) by BQ788, an ET-B receptor antagonist. Luteal cells were cultured for 24 h with medium alone (CTR), ET-3 (10⫺7 mol/L), or BQ788 (10⫺6 mol/L) or were preincubated for 30 min with increasing concentrations of BQ788 (10⫺9–10⫺6 mol/L) and then treated with ET-3 (10⫺7 mol/L). Data represent the mean ⫾ SEM of six experiments. Significance vs. CTR: *, P ⬍ 0.05; ***, P ⬍ 0.001. Significance vs. ET-3: E, P ⬍ 0.05; EE, P ⬍ 0.01.

FIG. 5. A, Stimulatory effect of cAMP and PMA on PGF2␣ levels measured in incubation medium of human luteal cells. The latter were cultured for 24 h with PMA (100 ng/mL) or cAMP (1 mmol/L) alone or with ET-1 (10⫺7 mol/L) and ET-3 (10⫺7 mol/L). Data represent the mean ⫾ SEM of six experiments. Significance vs. CTR: *, P ⬍ 0.05; ***, P ⬍ 0.001. Significance vs. ETs: EEE, P ⬍ 0.001. Significance vs. cAMP: §, P ⬍ 0.05. B, Stimulatory effect of cAMP and PMA on PGE2 levels measured in incubation medium of human luteal cells. The latter were cultured for 24 h with PMA (100 ng/mL) or cAMP (1 mmol/L) alone or with ET-1 (10⫺7 mol/L) and ET-3 (10⫺7 mol/L). Data represent the mean ⫾ SEM of six experiments. Significance vs. CTR: ***, P ⬍ 0.001. Significance vs. ETs: EEE, P ⬍ 0.001. Significance vs. cAMP: §, P ⬍ 0.05.

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creasing evidence indicates ETs as local modulators of ovarian function, as their presence and effect as well as the presence of their receptors have been demonstrated in both human and animal ovaries (4, 15, 25–27). We have recently demonstrated that ET-1 mRNA is expressed in human luteal cells, and that the peptide is able to negatively affect both basal and hCG-induced P synthesis, whereas ET-3 had no effect (8, 9). Our data are in keeping with recent findings showing a negative correlation between ET-1 and P in pooled samples of human follicular fluid (28). PGs are closely related to the luteal function. Human CL produces both PGE2 and PGF2␣ (29, 30). In various species, including humans, PGF2␣ seems to be of importance for luteolysis (20, 31–34), whereas there is increasing evidence of luteotropic activity exerted by PGE2. Within this frame, the present study was designed to continue our ongoing research about the effects of ETs on CL in an attempt to discover the novel action mechanism(s) by which ETs influence luteal physiology. We found that both ET-1 and ET-3 are able to significantly stimulate PGF2␣ and PGE2 synthesis and release from human luteal cells in a doseand time-dependent manner. For both PGs, the effects of ETs were observed at concentrations suggesting a physiological, rather than a pharmacological, action of the peptides. In addition, it is unlikely that these effects are secondary to a possible mitogenic action of ETs, as luteal cells were highly differentiated, and no mitosis was observed during the culture period (35). The observed stimulatory effect of ETs on PGE2 release was unexpected. In fact, based on the inhibition exerted by ET-1 on P production (9) and the fact that ET-1 mRNA expression is increased during the late luteal phase, when PGE2 levels are lowest (26, 36), we were postulating that the negative effect of ET-1 on P production might be mediated by an increase in PGF2␣ and a decrease in luteotropic PGE2 production, respectively. The fact that our results did not confirm our hypothesis might be explained in two ways. 1) PGs might influence the steroidogenic activity of CL in part through well established, classical ways and in part through alternative, as yet poorly understood mechanisms; this assumption seems to be confirmed by the fact that in the present study ET-3 has been shown to stimulate the synthesis of both PGs, whereas it had no effect on P production in the same experimental paradigm (9). 2) An alternative, or additional, hypothesis involves the vascular component of CL. Endothelial cells are particularly abundant in CL (⬎50% of total CL cells) (37, 38), where they are in contact with steroidogenic cells (39). Furthermore, endothelial cells produce ETs. It has been demonstrated that PGE2 inhibits ET-1 production and secretion from cultured bovine aortic endothelial cells (40), whereas PGF2␣ increases ET-1 content and inhibits P production in bovine CL slices (22). If these mechanisms are also present in human CL, we can hypothesize the presence of a feedback loop within the CL, where ETs and PGs mutually modulate each other’s production and action. Interestingly, both ETs and PGs can play a dual role: on steroidogenic cells and on arterial tone. Indeed, PGE2 has a luteotropic action (19) and is a potent vasodilator, whereas

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ET-1 inhibits P synthesis and is the most potent vasoconstrictor known (1). Conflicting are data regarding the location and type of ET receptors in the ovary. Several studies have demonstrated the presence of ET-A receptors in bovine luteal cells (22). In contrast, porcine and rat granulosa cells contain ET-B receptors, characterized by binding studies and mRNA expression (27, 41, 42). As far as the human ovary is concerned, Macina and colleagues (43) reported that ET-A and ET-B receptors are mostly expressed in the blood vessel of the ovary, rather than in the tissue. On the contrary, another group showed the presence of both receptor subtypes in luteinized granulosa cells, with the expression of the ET-A receptor mRNA being much greater than that of ET-B (44). In the present study we did not perform binding or molecular biology studies; however, the high degree of purity of our luteal cultures and the established selectivity of pharmacological tools indicate, albeit indirectly, that both ET receptor subtypes are expressed in cultured human luteal cells. In addition, findings with the antagonists of both ET receptors suggest that the action of ET-1 on PG release is mostly mediated by the ET-A receptor, whereas the effect of ET-3 is mediated by the ET-B subtype. The latter statement is based on the assumption that if the effects of ET-1 and ET-3 were in part mediated by both receptor subtypes, in both cases we could not have achieved complete inhibition of ET’s effects by blocking a single subtype. Our findings in experiments with cAMP and phorbol esters provide some additional evidence to the concept that in the ovary the ET-A receptor is coupled to the activation of phospholipase C via G protein (11–13, 44), whereas ET-B receptors signal via the adenylyl cyclase pathway (15). When used alone, cAMP and PMA stimulated PG release more strongly compared with ETs alone, and PMA was more effective than cAMP, probably because the protein kinase C pathway, activated by PMA, stimulates phospholipase A2, which, in turn, triggers the enzymatic cascade ending in PG biosynthesis. When ETs were added to the cultures containing PMA or cAMP, we found that ET-1 was able to further increase cAMP-induced PG release, whereas ET-3 had no such effect. These results might be explained assuming that ET-1, but not ET-3, activates the phospholipase C-protein kinase C pathway, which synergizes with the adenylyl cyclase pathway to produce maximal PG release. Conversely, the lack of potentiation by ET-3 suggests that this peptide signals through the adenylyl cyclase pathway, which is already maximally stimulated by cAMP and therefore is not capable of further increase. Although specifically exploring second messenger mechanisms, these experiments lend further support to the concept that in human luteal cells, ET-1 acts mainly via the activation of ET-A receptors, whereas the ET-B subtype mediates the effect of ET-3. In conclusion, we report that ET-1 and ET-3 are able to stimulate PG synthesis and release by human luteal cells. Taken collectively, the present data and our previous findings showing the presence of ET-1 mRNA in these cells as well as the ability of ET-1 to influence luteal steroidogenesis strongly support the idea that ETs play a primary role in CL physiology.

ETs AFFECT PG SYNTHESIS IN HUMAN LUTEAL CELLS References 1. Yanagisawa M, Kurihara H, Kimura S, et al. 1988 A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 332:411– 415. 2. Inoue A, Yanagisawa M, Kimura S, et al. 1989 The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci USA. 86:2863–2867. 3. Kimura S, Kasuia Y, Sawamura T, et al. 1988 Structure-activity relationships of endothelin: importance of the C-terminal moiety. Biochem Biophys Res Commun. 156:1182–1186. 4. O’Reilly G, Charnock-Jones DS, Davenport AP, et al. 1992 Presence of messenger ribonucleic acid for endothelin-1, endothelin-2 and endothelin-3 in human endometrium and a change in the ratio of ETA and ETB receptor subtype across the menstrual cycle. J Clin Endocrinol Metab. 75:1545–1549. 5. Kamada S, Kubota T, Taguchi M, Aso T. 1993 High levels of immunoreactive endothelin-1 in human follicular fluids. Hum Reprod. 8:674 – 677. 6. Magini A, Granchi S, Orlando C, et al. 1996 Expression of endothelin-1 gene and protein in human granulosa cells. J Clin Endocrinol Metab. 81:1428 –1433. 7. Kamada S, Blackmore PF, Kubota T, et al. 1995 The role of endothelin-1 in regulating human granulosa cell proliferation and steroidogenesis in vitro. J Clin Endocrinol Metab. 80:3708 –3714. 8. Apa R, Miceli F, de Feo D, et al. 1998 Endothelin-1: expression and role in Human corpus luteum. Am J Reprod Immunol. 40:370 –376. 9. Apa R, Miceli F, de Feo D, Mastrandrea ML, et al. 1998 Endothelin-1 inhibits basal and human chorionic gonadotrophin-stimulated progesterone production. Hum Reprod. 13:2425–2429. 10. Hosoda K, Nakao K, Hiroshi-Arai S, et al. 1991 Cloning and expression of human endothelin-1 receptor cDNA. FEBS Lett. 287:23–26. 11. Arai H, Hori S, Aramori I, et al. 1990 Cloning and expression of a cDNA encoding an endothelin receptor. Nature. 348:730 –732. 12. Sakurai T, Yanagisawa M, Takuwa Y, et al. 1990 Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature. 348:732–735. 13. Takuwa Y, Takuwa Y, Yanagisawa M, et al. 1989 A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts. J Biol Chem. 264:7856 –7861. 14. Takuwa Y, Kasuya Y, Takuwa N, et al. 1990 Endothelin receptor coupled to phospholipase C via a pertussin toxin-insensitive guanine vascular smooth muscle cells. J Clin Invest. 85:653– 658. 15. Iwai M, Hasegawa M, Taii S, et al. 1991 Endothelins inhibit luteinization of cultured porcine granulosa cells. Endocrinology. 129:1909 –1914. 16. Weems CW, Reynolds LP, Huie JM, Hoyer GL, Behrman HR. 1985 Effect of prostaglandin E1 or E2 on luteal function and binding of luteinizing hormone in non pregnant ewes. Prostaglandins. 29:161–164. 17. Vijayakumar R, Walters WA. 1983 Human luteal tissue prostaglandins, 17␤estradiol and progesterone in relation to the growth and senescence of the corpus luteum. Fertil Steril. 39:298 –303. 18. Dennefors B, Sjogren A, Hamberger L. 1982 Progesterone and 3⬘,5⬘-monophosphate formation by isolated corpora lutea of different ages. Influence of human chorionic gonadotropin and prostaglandin. J Clin Endocrinol Metab. 55:102–108. 19. Marsh JM, Lemaire WJ. 1974 Cyclic AMP accumulation and steroidogenesis in the human corpus luteum: effect of gonadotropins and prostaglandins. J Clin Endocrinol Metab. 38:99 –105. 20. Hahlin M, Dennefors B, Johanson C, Hamberger L. 1988 Luteotropic effects of prostaglandin E2 on the human corpus luteum of the menstrual cycle and early pregnancy. J Clin Endocrinol Metab. 66:909 –914. 21. Vijayakumar R, Walters WA. 1987 Ovarian stromal and luteal tissue prostaglandins, 17␤-estradiol and progesterone in relation to the phases of the menstrual cycle in women. Am J Obstet Gynecol. 156:947–951. 22. Bennegard B, Hahlin M, Wennberg E, Nore´n H. 1991 Local luteolytic effect of prostaglandin F2␣ in the human corpus luteum. Fertil Steril. 56:1070 –1076. 23. Apa R, Di Simone N, Ronsisvalle E, Miceli F, et al. 1996 Insulin like growth

24.

25.

26.

27.

28.

29.

30.

31.

32. 33.

34.

35. 36. 37. 38. 39.

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42. 43.

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factor (IGF)-I and IGF-II stimulate progesterone production by human luteal cells: role of IGF-I as mediator of growth hormone action. Fertil Steril. 66:235–239. Ciabattoni G, Pugliese F, Spaldi M, Cinotti GA, Patrono C. 1979 Radioimmunoassay measurement of prostaglandin E2 and F2␣ in human urine. J Endocrinol Invest. 2:173–179. Girsh E, Milvae RA, Wang W, Meidan R. 1996 Effect of endothelin-1 on bovine luteal cell function: role in prostaglandin F2␣-induced antisteroidogenic action. Endocrinology. 173:1306 –1312. Girsh E, Wang W, Mamluk R, et al. 1996 Regulation of endothelin-1 expression in the bovine corpus luteum: elevation by prostaglandin F2␣. Endocrinology. 137:5191–5196. Tedeschi C, Lohman C, Hazum E, et al. 1994 Rat ovarian granulosa cell as a site of endothelin reception and action: attenuation of gonadotropin-stimulated steroidogenesis via perturbation of the A-kinase signaling pathway. Biol Reprod. 51:1058 –1065. Plonowski A, Kaplinski AK, Radzikowska M, Borowiec M, Baranowska B. 1999 Correlation between 21 amino acid endothelin, intrafollicular steroids and follicle size in stimulated cycles. Hum Reprod. 14:2323–2327. Pathwardhan VV, Lanthier A. 1985 Luteal phase variations in endogenous concentrations of prostaglandins PGE and PGF and in the capacity for their in vitro formation in the human corpus luteum. Prostaglandin. 30:91–98. Challis JRG, Calder AA, Dilley S, et al. 1976 Production of prostaglandin E and F by corpora lutea, corpora albicantes and stroma from the human ovary. J Endocrinol. 68:401– 407. Csapo Al, Pulkkinen MO, Wiest WG. 1973 Effects of lutectomy and progesterone replacement therapy in early pregnant patients. Am J Obstet Gynecol. 115:759 –765. Horton EW, Poyser NL. 1976 Uterine luteolytic hormone: a physiological role for prostaglandin F2␣. Physiol Rev. 56:595– 601. Korda AR, Shutt DA, Smith ID, Shearman RP, Lyneham RC. 1975 Assessment of possible luteolytic effect of intraovarian injection of prostaglandin F in the human. Prostaglandin. 9:443– 447. Pathwardhan VV, Lanthier A. 1984 Effect of prostaglandin F2␣ on the hCGstimulated progesterone production by human corpora lutea. Prostaglandins. 27:465– 470. Retamales I, Carrasco I, Troncoso JL. 1994 Morphofunctional study of human luteal cell subpopulation. Hum Reprod. 9:591–596. Usuki S, Suzuki N, Matsumoto H, et al. 1991 Endothelin-1 in luteal tissue. Mol Cell Endocrinol. 80:147–151. Rodgers RJ, O’Shea JD, Bruce NW. 1984 Morphometric analysis of the cellular composition of the ovine corpus luteum. J Anat. 138:757–769. O’Shea JD, Rodgers RJ, D’Occhio MJ. 1989 Cellular composition of cyclic corpus luteum of the cow. J Reprod Fertil. 85:483– 487. Girsh E, Greber Y, Meidan R. 1995 Luteotrophic and luteolytic interactions between bovine small and large luteal-like cells and endothelial cells. Biol Reprod. 52:954 –962. Prins BA, Hu R-M, Nazario B, Pedram A, Frank HJL, Weber MA, Levin ER. 1994 Prostaglandin E2 and prostacyclin inhibit the production and secretion of endothelin from cultured endothelial cells. J Biol Chem. 22:11938 –11944. Iwai M, Hori S, Shigemoto R, Kanzaki H, Mori T, Nakanishi S. 1993 Localization of endothelin receptor messenger ribonucleic acid in the rat ovary and fallopian tube by in situ hybridization. Biol Reprod. 49:675– 680. Kubota T, Kamada S, Aso T. 1994 Endothelin-1 as a local ovarian regulator in porcine granulosa cells. Horm Res. 41(Suppl):29 –35. Macina R, Barni T, Calogero A, et al. 1997 Identification, characterization and biological activity of endothelin receptors in human ovary. J Clin Endocrinol Metab. 82:4122– 4129. Kamada S, Blackmore PF, Kubota T, et al. 1995 The role of endothelin-1 in regulating human granulosa cell proliferation and steroidogenesis in vitro. J Clin Endocrinol Metab. 80:3708 –3714.