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Angiotensin-(1–7): A Novel Peptide in the Ovary

Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais, 31270-901 Belo Horizonte, Brazil

Address all correspondence and requests for reprints to: Dr. Adelina Martha dos Reis, Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Antonio Carlos 6627, CEP 31270–901, Belo Horizonte MG, Brazil. E-mail: adelina{at}icb.ufmg.br.

	Abstract

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The present study was undertaken to investigate the presence of angiotensin-(1–7) [Ang-(1–7)] in the ovary and a possible role for it. Cycling female rats were killed in each phase of the estrous cycle, and ovarian Ang II and Ang-(1–7) were separated by HPLC and measured by RIA. The mean levels of Ang-(1–7) in proestrus and estrus were significantly higher than those in metestrus and diestrus (P < 0.05). Ang-(1–7) was also significantly higher in equine chorionic gonadotropin (eCG)-treated immature rats. Ang-(1–7) induced a significant increase in estradiol and progesterone production (P < 0.05) in the ovary of immature rats (24–25 d old) pretreated with eCG and perfused in a closed circuit system. This effect was blocked by A-779, a specific Ang-(1–7) antagonist (P < 0.05). The present data demonstrate the presence and physiological role of a novel renin-Ang system peptide in the ovary. The higher level of Ang-(1–7) in proestrus and estrus as well as in eCG-treated rats suggests the involvement of this renin-Ang system peptide in pre- and postovulatory events.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE IDENTIFICATION of prorenin, renin, angiotensinogen, angiotensin-converting enzyme (ACE), angiotensin II (Ang II), and Ang II receptors in the ovary suggests that there is a functional ovarian renin-Ang system (RAS). It may play a significant role in several areas of ovarian physiology, such as follicular development, steroidogenesis, oocyte maturation, ovulation, and follicular atresia (reviewed by in Ref. 1). Nonetheless, most studies of ovarian RAS have been performed in rodents (1), a number of relevant experiments have also been carried out in bovines and humans (2, 3), and functional roles for ovarian RAS in development, ovulation, and luteal function have been suggested (3). Disturbances in the ovarian RAS can be the cause or the consequence of serious reproductive disorders, such as polycystic ovary syndrome and ovarian hyperstimulation syndrome (4, 5, 6, 7).

However, despite the large number of studies of ovarian RAS, understanding of the physiological role of Ang II in some events has been impaired by discrepant findings. Saralasin, a nonspecific Ang receptor antagonist, blocked ovulation in immature rats treated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) (8) and inhibited hCG-induced ovulation in rabbit ovaries perfused in vitro (9, 10). In contrast, an autoradiographic study demonstrated the presence of great number of Ang II receptors in atretic follicles (11), but not in preovulatory follicles containing LH receptors (12). Similarly, inhibition of Ang II production using captopril, an ACE inhibitor, did not affect ovulation in immature rats in which ovulation was induced with eCG and hCG or in perfused rabbit ovaries, suggesting that locally produced Ang II is not essential for processes that affect ovulation (13, 14, 15). Potential mechanisms for explaining the lack of a captopril effect on ovulation include the generation of other Ang fragments and different pathways for Ang II production without ACE participation. Ang-(1–7), a biologically active member of the RAS that can be formed by an ACE-independent pathway, could play a role in ovulation and ovarian regulation. This Ang is the most pleiotropic of the Ang II derivatives, producing effects similar to, opposite from, or distinct from those of Ang II [reviewed by Santos et al. (16)]. Ang-(1–7) can be generated by processing of Ang I by endopeptidases (NEP 24.11 and prolyl-endopeptidase) or of Ang II by prolyl-endopeptidase or prolyl-carboxy-peptidase and ACE-2 [reviewed by Santos et al. (17)]. Although a specific receptor for Ang-(1–7) has not yet been cloned, evidence is accumulating for the existence of a selective Ang-(1–7) receptor, especially after the availability of its selective antagonist D-Ala⁷-Ang-(1–7) (A-779; Ref. 18).

The purpose of this study was to determine the presence of Ang-(1–7) and its possible physiological role in the ovary. Our results show that Ang-(1–7) is present in the rat ovary, that its concentrations vary during the estrous cycle, and that it induces estrogen production in perfused ovaries of eCG-treated rats.

	Materials and Methods

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Animals
Female Wistar rats obtained from CEBIO-UFMG (Belo Horizonte, Brazil) were cared for according to the international guidelines for animal care. The experimental protocol was approved by the ethics committee in animal experimentation of Federal University of Minas Gerais. The animals (four or five per cage) were maintained under controlled light and temperature conditions (lights on from 0500–1900 h; 23 ± 3 C) and had free access to tap water and a standard rat chow (Nuvital Nutrientes Ltd., Colombo, Brazil). For determination of Angs in the ovaries, adult rats, weighing 220–250 g, were used. After at least three consecutive regular estrous cycles, the animals were killed by decapitation between 1000–1100 h, and the ovaries were pooled into four groups according to the cycle phase. Immature rats (24–25 d old) injected sc with eCG (20 IU in 0.2 ml; Sigma-Aldrich Corp., St. Louis, MO) 48 h before the experiments were used for ovarian Ang-(1–7) determination and for ovarian perfusion experiments.

Ovarian processing for determination of Ang concentrations in rat ovaries
After decapitation, the ovaries were immediately removed, cleaned of fat, frozen in liquid nitrogen, and stored at -80 C until processing. Pairs of ovaries were then homogenized in 0.1 N acetic acid solution containing the following protease inhibitors: 10^-5 M phenylmethylsulfonylfluoride, 10^-5 M pepstatin A, 10^-5 M EDTA, 10^-5 M p-hydroxymercuribenzoate, and 9 x 10^-4 M orthophenanthroline, all purchased from Sigma-Aldrich Corp. After centrifugation (20,000 x g, 4 C, 30 min), peptides were extracted using Sep-Pak C₁₈ cartridges (Waters Corp., Milford, MA) activated by sequential washes with acetonitrile and ammonium acetate (pH 4) and eluted with acetonitrile/ammonium acetate solution (99:1). The samples were dried in a SpeedVac (Eppendorf concentrator, Hamburg, Germany) apparatus and stored at -80 C until processing.

Determination of ovarian Ang
Ang peptides were analyzed by a combination of HPLC and RIA (19, 20, 21, 22). HPLC was performed on a Shimatzu HPLC system (Shimatzu Co., Kyoto, Japan) equipped with two LC-9A pumps controlled by a SCL-6B system controller, an SPD-6A UV-Vis spectrophotometric detector, and a C-R6A chromatopic integrator. Reverse separation was carried out using a Vydac RP-18 column (25 x 0.46 cm; particle size, 5 µm; Vydac, Hesperia, CA) coupled to a Vydac guard column. Gradient elution was obtained with aqueous 0.13% Hepta-fluor-butiric acid (mobile phase A) and 0.13% Hepta-fluor-butiric acid/80% acetonitrile in water (mobile phase B). Gradient conditions were 30–45% mobile phase B, in 40 min, 62–90% B from 50–60 min, 100% B from 62–64 min, and 62% to 30% B from 65–70 min at a flow rate of 1.0 ml/min. Samples were collected every 30 sec and evaporated in a vacuum centrifuge (Eppendorf, Brinkmann Instruments, Inc., West Orange, NY) before RIA. Calibration of the system was achieved by injection of cold synthetic Angs and Ang fragments or ¹²⁵I-labeled Angs, with each sample monitored by a -counter or assayed by RIA. HPLC fractions 1–32 were assayed for Ang-(1–7) immunoreactivity (-ir), fractions 33–62 were processed for Ang II-ir, and fractions 63–95 were assayed for Ang I-ir. The HPLC grade solvents were obtained from Merck \|[amp ]\| Co., Inc. (Darmstadt, Germany). The standard Ang peptides were purchased from Bachem (Torrance, CA).

Calibration of the HPLC column, using a mixture of synthetic standards, revealed a clearly defined sequence of the retention times. For detection of different peptides, three specific antibodies were used: one for Ang-(1–7); one that recognizes Ang-(4–8), Ang-(3–8), Ang II, and Ang-(2–8); and one that recognizes Ang-(3–10), Ang-(4–10), Ang I, and Ang-(2–10). Figure 1 shows that under our conditions these peptides were resolved as clearly identifiable peaks.

View larger version (24K): [in this window] [in a new window]   Figure 1. Chromatographic separation of a mixture of synthetic Ang peptides by reverse phase HPLC (see text for chromatographic conditions). The mixture of peptides was fractionated by HPLC and detected by RIA. Three different antibodies were used for measurement of the peptides by RIA: one for Ang-(1–7); another for Ang-(4–8), Ang-(3–8), Ang II, and Ang-(2–8); and another for Ang-(4–10), Ang I, and Ang-(2–10).

Ovarian perfusion
The surgical procedure for rat ovary isolation was previously described by Koos et al. (23). Although the details of the standard perfusion system have been modified, the basic components have remained constant. The immature rats were anesthetized with sodium pentobarbital (30 mg/kg) and received 500 IU/100 g heparin sulfate, ip (Liquemine, Roche, Rio de Janeiro, Brazil). After laparotomy, the aorta artery was cannulated close to the right ovarian artery bifurcation. All branches of the aorta and vena cava were ligated and severed. The right uterus-ovarian artery, vein, and tip of the uterine horn were doubly ligated en masse just adjacent to the ovary and oviduct and severed. Aorta and vena cava were ligated en masse near the bifurcation and severed. The right ovary preparation was then removed from the animal and perfused with cold saline solution to completely wash out blood. Finally, the ovarian preparation was attached to the perfusion apparatus via the aortic cannula.

The perfusion system was similar to that described by Brännström et al. (24) and consisted of a closed system in which the medium recirculates. The medium consisted of 40 ml medium 199 with Earle’s salt, L-glutamine (Sigma-Aldrich Corp.) supplemented with 4% BSA (Sigma-Aldrich Corp.), 18 mM HEPES (Sigma-Aldrich Corp.), 0.02 µg/ml insulin (Biohulin, Biobras, Montes Claros, Brazil), 0.2 µg/ml heparin sulfate (Liquemine Roche), and 60 µg/ml gentamicin sulfate (Garamicina, Schering, Rio de Janeiro, Brazil). Temperature (37 C), pH (7.4), pressure (80 mm Hg), and oxygenation (95% O₂/5% CO₂) were maintained constant (modified from Ref. 23). All tubes and glass components of the perfusion system were siliconized to avoid steroid adherence to the walls.

Samples of perfusion medium (1.8 ml) were taken at 0, 1, 2, 3, 4, 6, 8, and 10 h after the 1-h stabilization period. The peptides, Ang II (1 µM), Ang-(1–7) (1 µM), and A-779 (1 µM) were added to the perfusion medium immediately after the first sample collection (0 h). The volume removed was replaced with the same volume of fresh medium containing the peptide. Samples were frozen at -20 C until processing.

Estradiol and progesterone RIA
Estradiol and progesterone concentrations in the perfusion medium were determined by RIA. After extraction with ethyl ether, the perfusion medium was dried and reconstituted in assay buffer. Estradiol and progesterone antibodies produced in rabbits and donated by Dr. José Antunes Rodrigues (FMRP-USP, Ribeirão Preto, Brazil) were used for RIA. Estradiol and progesterone standards (purchased from Sigma-Aldrich Corp.) and [³H]estradiol and [³H]progesterone (Amersham Pharmacia Biotech, Arlington Heights, IL) were used. Separation was performed with 200 µl of a suspension consisting of 625 mg activated charcoal (Merck \|[amp ]\| Co., Inc.) and 62.5 mg dextran (D-1537, Sigma-Aldrich Corp.) in gel/0.1% phosphate buffer. The radioactivity was counted in a scintillation analyzer ß-counter (TR-1600, Packard Instrument Co., Meridian, NJ), using a scintillation cocktail consisting of 5 g 2,5-diphenyloxazole (Merck \|[amp ]\| Co., Inc.) and 625 mg 1,4-bis[5-phenyl-2-oxazolyl]benzene/2,2'-p-phenylene-bis[5-phenyloxazole] (Sigma-Aldrich Corp.) in toluene/methanol (980/20 ml; Merck \|[amp ]\| Co., Inc.).

Statistical analysis
Data were expressed as the mean ± SEM. Ovarian Ang concentrations were analyzed by the Kruskal-Wallis test. Estradiol and progesterone concentrations in the perfusion medium were analyzed by two-way ANOVA, followed by the Newman-Keuls test. Comparisons with P < 0.05 were considered significant.

	Results

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Ang-(1–7), Ang II, and Ang I levels in rat ovary
Figure 2 displays a representative reverse phase HPLC profile of ovarian Ang-(1–7), Ang II, and Ang I immunoreactivity during different phases of the rat estrous cycle. Three distinct peaks were identified by specific antibodies for Ang-(1–7), Ang II, and Ang I, respectively. Ovarian levels of Ang-(1–7) were higher in proestrus (Fig. 2A) and estrus (Fig. 2B) than in metestrus (Fig. 2C) and diestrus (Fig. 2D). The ovarian levels of Ang II were higher in proestrus, lower in metestrus and diestrus, and undetectable in estrus in this particular experiment. Ang I-ir was present in all estrous cycle phases.

View larger version (24K): [in this window] [in a new window]   Figure 2. Representative reverse phase HPLC profile of ovarian Ang-(1–7), Ang II, and Ang I-ir during different phases of the rat estrous cycle (A, proestrus; B, estrus; C, metestrus; D, diestrus). Three distinct peaks were identified by specific antibodies for Ang-(1–7), Ang II, and Ang I, respectively.

View larger version (13K): [in this window] [in a new window]   Figure 3. Concentrations of Ang-(1–7) (A) and Ang II (B) in rat ovary in different phases of estrous cycle. The data show the mean ± SEM of three pools of ovaries in each phase of the estrous cycle. Ang-(1–7) and Ang II were measured by RIA after HPLC processing. Different letters denote statistically different values (P < 0.05).

Isolated perfused ovaries
The physiological role of Ang-(1–7) was tested using perfused ovaries of immature rats pretreated with eCG. Ang-(1–7) 10^-6 M induced a significant increase in estradiol (Fig. 4A) and progesterone (Fig. 5A) production starting at the first hour and increasing up to the sixth hour of perfusion. The effect of Ang II (Figs. 4B and 5B) was similar to that of Ang-(1–7).

View larger version (16K): [in this window] [in a new window]   Figure 4. Effects of Ang-(1–7) (A) and Ang II (B) on estradiol production by perfused rat ovary. Ovaries of immature rats pretreated 48 h previously with eCG (20 IU) were perfused with medium alone, 1 µM Ang-(1–7), 1 µM Ang II, or a combination of Ang-(1–7) and A-779 (A) or Ang II and A-779 (B). Data points show the mean ± SEM of six or seven ovary perfusions. *, P < 0.05 vs. perfusion with medium alone; **, P < 0.05 vs. Ang-(1–7).

View larger version (16K): [in this window] [in a new window]   Figure 5. Effects of Ang-(1–7) (A) and Ang II (B) on progesterone production by perfused rat ovary. Ovaries of immature rats pretreated 48 h before with eCG (20 IU) were perfused with medium alone, 1 µM Ang-(1–7), 1 µM Ang II, or a combination of Ang-(1–7) and A-779 (A) or Ang II and A-779 (B). Data points show the mean ± SEM of five to seven ovary perfusions. *, P < 0.05 vs. perfusion with medium alone; **, P < 0.05 vs. Ang-(1–7) or Ang II.

A-779 also blocked the effect of Ang-(1–7) on progesterone production (Fig. 5A). A significant decrease in progesterone production was observed in A-779-perfused ovaries compared with controls (P < 0.05; data not shown).

To verify whether the effect of Ang II on steroidogenesis was due to its conversion to Ang-(1–7), ovaries were perfused with a combination of Ang II and A-779. No inhibition of estradiol production was observed (Fig. 4B), indicating that the two peptides act through different receptors. However, A-779 blocked the progesterone production induced by Ang II (Fig. 5B), indicating that Ang-(1–7) could be involved in this response.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study provides the first evidence that Ang-(1–7), a biologically active member of the RAS, is present in the rat ovary and that its level varies during the estrous cycle. Ovarian Ang-(1–7) levels are maximal at proestrus and estrus, and Ang-(1–7) induces estradiol production, an effect that is blocked by its specific antagonist, A-779. These findings raise the possibility that Ang-(1–7) plays a role in preovulatory and immediate postovulatory events.

The ovarian concentrations of Ang-(1–7) at proestrus and estrus and in eCG-treated immature rats were higher than the plasma concentrations determined in our laboratory for male Wistar rats (21, 22) and were also higher than those in other organs, such as heart, kidney, and adrenal, of female Sprague Dawley rats (25). Senanayake and co-workers (25) demonstrated that plasma Ang-(1–7) increased during proestrus, but the circulating levels of Ang II were unaffected by the estrous cycle. Ang-(1–7) can be formed from Ang II by Ang II hydrolysis by prolyl-endopeptidase (PEP) or prolyl-carboxypeptidades or directly from Ang I by NEP 24.11 or PEP (16). To our knowledge, the presence of NEP in the rat ovary has not been demonstrated. However, in the uterus the level of NEP is increased by progesterone and decreased by estrogen (26). Considering the high levels of estrogen production in the proestrous ovary, NEP does not seem to be involved in the increase in Ang-(1–7) observed in proestrus. On the other hand, the levels of PEP in mouse ovaries were observed to be higher in estrus and lower in diestrus (27). Therefore, it is likely that PEP, rather than NEP, would be involved in the increase in Ang-(1–7) observed in proestrus and estrus and in eCG-treated rats. In addition, the possible involvement of the Ang-(1–7)-forming enzyme, ACE-2 (17), should be considered.

In this study we found the ovarian Ang II concentration to be more than 10-fold higher in proestrus than in any other phase of the estrous cycle. Also, these levels were higher than the plasma concentrations found in male Wistar rats (21, 22) and even higher than those observed in female Sprague Dawley rats (25). Husain et al. (28) also demonstrated that the levels of Ang II-ir were 8- to 65-fold higher in the rat ovary than in plasma. These levels were not changed by bilateral nephrectomy, indicating that ovarian Ang II content is not due to a simple contamination of this tissue with circulating Ang II. The possibility that the Ang II present in the ovary only represents the peptide sequestered by ovarian Ang II receptors was excluded by studies showing that, in addition to prorenin, renin, and ACE, angiotensinogen is present in the ovary (29). The demonstration of angiotensinogen mRNA in the ovary (30) provided further evidence to exclude the possibility that Ang II-ir in the ovary is merely derived from the bloodstream.

The angiogenic actions of Ang II have been demonstrated (31), and the high levels observed in the present study during proestrus could be involved in the vascular proliferation observed in this phase of the estrous cycle. Ang-(1–7), on the other hand, has been demonstrated to have antiproliferative and antiangiogenic effects (32, 33). Therefore, the differential presence of both peptides, Ang II present in proestrus and Ang-(1–7) in proestrus and estrus, could be part of a coordinated array of proliferative events that occurs during pre- and postovulatory phases of the estrous cycle.

Ang II is very well known for its vasoconstrictor action. However, no ovarian vasoconstriction is observed after Ang II treatment. Instead, vasodilatation was observed in rabbit ovaries perfused in vitro with Ang II (34). Although this effect could be accounted for by prostaglandin release induced by Ang II, other contraregulatory factors, such as natriuretic peptides, that usually play a role opposite that of the RAS as systemic regulators of blood pressure and fluid homeostasis cannot be discounted. The presence of natriuretic peptides, atrial natriuretic peptide and C-type natriuretic peptide, has been demonstrated in rat ovaries as well as their fluctuations throughout the estrous cycle; they reach a peak around midcycle at the time of ovulation (35, 36, 37). In addition, Ang-(1–7), instead of being a vasoconstrictor like Ang II, has been demonstrated in different species and preparations to have vasodilator properties (16) and could be another factor involved in the vasodilatation reported in the proestrus ovary.

In the present study, we used a well characterized model for in vitro perfusion of preovulatory rat ovaries using a medium devoid of any circulating factors that could interfere with estradiol production to examine the effects of angiotensins on steroidogenesis. Both Ang-(1–7) and Ang II significantly stimulated the production of estradiol and progesterone. Ang II has been shown to stimulate estradiol production in different species and preparations (10, 38, 39, 40, 41). The effect of Ang II on progesterone production is somewhat controversial. No effect was observed by others (38) in eCG-treated rats, probably because they used a shorter incubation time (3 h) than we did. In human granulosa cultured cells, both inhibitory and stimulatory effects of Ang II were observed depending on the time of culture, and these actions seemed to be mediated by AT2 receptors (41).

The observation that Ang II stimulates estradiol production is consistent with the localization of Ang II receptors on the granulosa cells of the developing ovarian follicles (42). Ovarian follicular estrogen levels are of fundamental importance in the selection and maintenance of dominant follicles (43, 44). The presence of Ang II receptors on the developing follicles in the ovary (28), together with the selective effects of Ang II on estrogen secretion, suggests a role for Ang II in maintaining intrafollicular estrogen levels during follicular maturation. The signal transduction mechanism coupled to the steroidogenic effect of Ang II is not clear. However, in granulosa cells from hypophysectomized, diethylstilbestrol-treated rats, Ang II had no effect on basal or stimulated phosphoinositide turnover, intracellular calcium levels, or cAMP production (45).

The effect of Ang-(1–7) on the production of estradiol and progesterone was as potent as that of Ang II. This effect was blocked by A-779, a selective antagonist, indicating that a specific Ang-(1–7) receptor is involved in this response. To our knowledge, this is the first demonstration of a steroidogenic effect of Ang-(1–7). The inhibition of basal progesterone production by A-779 seems to indicate an action of endogenous Ang-(1–7). In contrast to Ang II, which has been extensively studied, the localization of Ang-(1–7) in the ovary has not been described. We did not investigate the signal transduction mechanism coupled to the steroidogenic action of Ang-(1–7). It has been shown that Ang-(1–7) stimulates the release of prostaglandins from porcine aortic endothelial cells as well as vascular smooth muscle cells (46, 47). Prostaglandins are involved in the ovulation induced by the increase in Ang II concomitantly with estradiol production (10). Thus, it is likely that prostaglandins may be involved in the effects of ovarian RAS in the maintenance of intrafollicular estrogen levels in the preovulatory follicles.

As Ang-(1–7) and Ang II had similar effects on estradiol and progesterone production, the effect of Ang II could be due to its conversion to Ang-(1–7). Ovary perfusion using a combination of Ang II and A-779 resulted in no inhibition of estradiol production. This result indicates that the two peptides must be acting through different receptors: Ang-(1–7) receptors antagonized by A-779 and Ang II receptors not antagonized by A-779. On the other hand, the effect of Ang II on progesterone production was blocked by A-779. Therefore, it seems that the effect of Ang II is due to its conversion to Ang-(1–7). The localization, mechanism of action, and other roles of Ang-(1–7) in the ovarian physiology remain to be determined, but it seems likely that Ang II and Ang-(1–7) could play complementary roles.

It has been recently reported that female mice lacking angiotensinogen have normal fertility (48). This is not surprising considering that the development of ovarian follicles is regulated by a vast array of hormones and growth factors, but the key role in the process is played by gonadotropins and gonadal steroids. Thus, RAS could be considered one of the modulators of ovarian physiology. However, as serious disturbances of the reproductive process, such as polycystic ovary syndrome and ovarian hyperstimulation syndrome, have been associated with aberrations of RAS (4, 5, 6, 7), this modulatory role seems to be very important.

In summary, the present results indicate that Ang-(1–7) may play a role in pre- and postovulatory events. To our knowledge, this is the first report showing that Ang-(1–7) is produced in a cyclic manner and has a physiological role in the ovary.

	Acknowledgments

 
We thank Jacqueline Braga Pereira for her expert assistance with the RIA, and Drs. Jose Antunes Rodrigues and Ana Lucia V. Favaretto for their generous gift of the RIA antibodies. We also thank Dr. Fernando M. Reis for carefully reading the manuscript.

	Footnotes

 
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnólogico, Fapemig, and Pronex. Presented in part at the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000.

¹ A.P.R.C. is the recipient of a Coordenação de Aperfeiçoamento de Pessoal de Nível Superior scholarship.

² Present address for A.P.R.C.: Department of Morphophysiology, Federal University of Piaui, Teresina, 64049-550.

Abbreviations: ACE, Angiotensin-converting enzyme; Ang, angiotensin; eCG, equine chorionic gonadotropin; hCG, human chorionic gonadotropin; -ir, immunoreactivity; NEP, neutral endopeptidase 24.11; PEP, prolyl-endopeptidase; RAS, renin-angiotensin system.

Received October 25, 2002.

Accepted for publication January 28, 2003.

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