Autoradiographic Localization and Characterization of Angiotensin II Receptors in the Bovine Placenta and Fetal Membranes¹

^a Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark

	ABSTRACT

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Autoradiography and angiotensin (Ang) II receptor binding studies showed that all parts of the bovine placenta and fetal membranes contained high densities of Ang II receptors throughout gestation. The receptors were predominantly subtype 2 (AT₂) receptors in the fetal and subtype 1 (AT₁) receptors in the maternal compartment. In the allantoamnionic membrane, Ang II receptors were evenly distributed in the mesenchymal tissue, with the highest expression around the few arteries. In the intercotyledonary and cotyledonary allantochorionic membrane, AT₂ receptors as well as the less-expressed AT₁ receptors were located on mesenchymal cells, especially adjacent to the allantoic endoderm, trophoblast cell layer, and arteries. In the mesenchymal tissue of the placentome, Ang II receptors were mostly expressed at the main branches of the fetal villi of the cotyledons. In the maternal part of the placentome, mainly AT₁ receptors but also low densities of AT₂ receptors and non-AT₁/non-AT₂ Ang II binding sites were found close to the stalk and at the main branches of the maternal crypts. Autoradiography revealed no changes in the pattern of distribution of the Ang II receptors throughout gestation. It is suggested that Ang II has an effect on regulatory as well as growth processes in these tissues.

	INTRODUCTION

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Angiotensin (Ang) II exerts a variety of physiological effects in the cardiovascular, endocrine, and nervous system when its specific receptors are stimulated [1]. A number of studies have provided evidence for the presence of all components of local tissue renin-angiotensin systems (RAS) in the uteroplacental unit in humans as well as in many other species. These studies strongly suggest that these local RAS have regulatory functions in the placenta [2, 3]. Ang II receptors comprise two main types, termed AT₁ and AT₂ receptors [4]. They are distinguished by their biochemical and pharmacological properties, including their differences in affinity for nonpeptide AT₁ antagonists such as losartan (DuP 753) and AT₂ antagonists such as PD 123319 [5, 6]. The existence of non-AT₁/non-AT₂ Ang II binding sites has previously been demonstrated [4]. Recently, such binding sites were also found in the human endometrium [7]. The known effects of Ang II in the uteroplacental unit have mainly been associated with the AT₁ receptor. In humans and in ewes, Ang II is one of the factors that regulate the blood flow in the uteroplacental unit, and it thereby indirectly influences the fetal volume homeostasis and oxygenation [8–10]. Ang II also stimulates placental lactogen, pregnancy-specific ß₁-glycoprotein, and estradiol secretion in human trophoblast cells via AT₁ receptors [11–13]. The physiological effects of the AT₂ receptor are presently much less characterized. It has been suggested, however, that the AT₂ receptor participates in the control of cell proliferation and/or differentiation [1]. The identity and roles of non-AT₁/non-AT₂ Ang II binding sites are unclarified. Species differences in Ang II receptor expression in the uteroplacental unit were previously demonstrated (for review see Nielsen et al. [2]). Recently, we demonstrated a predominance of AT₂ receptors in the porcine placenta and fetal membranes [14]. This was an exception to the finding that Ang II receptors are almost exclusively AT₁ receptors in the human placenta [15]. These results indicate species differences in the function of the RAS in the placenta. A further characterization of species differences in the expression and tissue distribution of Ang II receptors in the placenta and fetal membranes may provide a better understanding of the roles of the RAS in these tissues. The expression of Ang II receptors has never been studied before in the bovine uteroplacental unit. In the present study, we have investigated this by using Ang II receptor binding studies and autoradiography. We collected tissue from three functionally different areas illustrated in Figure 1a: A) the allantoamnionic membrane; B) the placentome, the region in which the allantochorionic membrane forms villous trees (cotyledons) that fit into the crypts formed by the maternal endometrium (caruncles), in which the fetal and maternal capillaries are in close relationship, and in which the uterine epithelium and the trophoblast cell layer remain intact (see enlarged drawing in Fig. 1b); and C) the smooth part of the placenta, where the allantochorionic membrane is apposed to the uterus and where the uterine milk is secreted.

View larger version (74K): [in this window] [in a new window]   FIG. 1. a) Schematic drawing of a bovine fetus in utero. A, allantoamnionic membrane; B, placentome; C, smooth part of the placenta. The white area represents the fetal membranes. IrC, intercotyledonary region. b) Detailed schematic drawing of a placentome (the box in a). The allantochorion (AC) is smooth at the IcR, which is apposed to the likewise smooth endometrium. In the placentome, the AC forms fetal villous trees (FV), cotyledons. The gray part represents the maternal compartment, which forms complementary crypt walls (CW), caruncles, in the placentomes. In the AC, a loose network of mesenchymal cells (MC) is covered with allantoic endoderm (AE) towards the allantoic cavity and with an intact trophoblast cell layer (TrC) apposing the uterine epithelium (UE). *Area of the placentome in which higher-order villi and crypts interdigitate, seen in Figures 7 and 8. a is reproduced from Hagemann et al., Clin Exp Pharmacol Physiol 1993; 20:41-50 [20], with permission. Drawings by Jesper Tom-Petersen.

	MATERIALS AND METHODS

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Collection of Tissues

Bovine placentae and fetal membranes were obtained at an abattoir 20–30 min after death. The stage of gestation was estimated by the fetal crown-rump length [16]. The animals used for Ang II receptor binding studies (n = 23) were grouped according to 3 stages of gestation: Days 1–95 (7 animals; median 75 days, range 40–92 days), Days 96–190 (9 animals; median 131 days, range 108–190 days), and Days 191–290 (7 animals; median 252 days, range 191–275 days). Tissue samples for Ang II receptor binding studies were transported to the laboratory on ice (1–2 h) and stored at -20°C. Tissue samples for autoradiography were collected from animals (n = 16) at different days of gestation ranging from Day 50 to 252, snap-frozen in liquid nitrogen, wrapped in Parafilm (American Can Company, Greenwich, CT) to prevent dehydration, transported on dry ice, and stored at -80°C.

Homogenization of Tissue and Preparation of Membrane Fractions

Cell membrane fractions for Ang II receptor binding studies were prepared by homogenization of 1–2 g of tissue in 10 ml of 50 mM Tris-HCl (pH 7.4) containing 10 mM EDTA (Merck, Darmstadt, Germany) and 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma, St. Louis, MO). The homogenization was performed at 0°C by using the dispersing instrument Ultra Turrax T8 (IKA Labortechnik, Staufen, Germany) at setting 3 for 30 sec followed by the Potter-Elvehjem homogenizer (B. Braun, Melsungen, Germany) for 3 min at 250 rpm. The supernatant, obtained by centrifugation of the homogenate at 600 x g for 10 min at 4°C, was centrifuged at 45 000 x g for 25 min at 4°C. The pellet was suspended in 10 mM sodium phosphate (pH 7.4) containing 120 mM sodium chloride, 1 mM EDTA (Merck), and 1 mM PMSF, centrifuged at 45 000 x g for 25 min at 4°C, and suspended in the same buffer with 5 mM MgCl₂. The cell membrane fractions were frozen at -20°C for later analysis.

For measurement of renin concentrations, 100 mg of tissue was homogenized at 0°C in 0.5 ml of 10 mM sodium phosphate (pH 7.5) containing 140 mM sodium chloride, 10 mM EDTA, 10 mM N-ethylmaleimide (Sigma), 2 mM 8-hydroxyquinoline (Merck), and 0.3 mM sodium azide. The Ultra Turrax T8 was used for 30 sec followed by the Potter-Elvehjem homogenizer for 3 min at 250 rpm. The homogenate was centrifuged at 1850 x g for 15 min at 4°C. The supernatant was stored at -20°C for later analysis.

Angiotensin II Receptor Binding Studies

The Ang II receptor assay was performed in duplicate at 37°C in a final volume of 150 µl. One hundred microliters of suspended cell membrane fraction, in which the cell membrane protein concentration varied between the various preparations, was incubated with 25 µl of ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II (specific activity 2200 Ci/mmol; Du Pont, NEN Research Products, Wilmington, DE) in 10 mM sodium phosphate (pH 7.4) containing 120 mM sodium chloride, 1 mM EDTA, 1 mM PMSF, 5 mM MgCl₂, 0.5 g/L soybean trypsin inhibitor, type I-S (Sigma), 0.6 g/L bacitracin (Sigma), and 2 g/L human serum albumin (Statens Seruminstitut, Copenhagen, Denmark). The final concentrations of ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II were 18-52 pM. For characterization of the receptor type, 25 µl of varying concentrations of losartan (DuP 753; Du Pont Merck Pharmaceutical Company, Wilmington, DE), or PD 123319 ((S)-1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid, ditrifluoroacetate, monohydrate; Parke-Davis, Ann Arbor, MI) in 0.2 M Tris-HCl (pH 7.5) containing 2 g/L human serum albumin were used as ligands. In this way, the relative amounts of the Ang II receptors that were AT₁ or AT₂ receptors were determined by displacement curves obtained with losartan and PD 123319 in a number of animals in each group. The density of Ang II binding sites and their binding characteristics for [Sar¹-Ile⁵-Ile⁸]-Ang II were determined by using 25 µl of varying concentrations (0.1–10 nM) of unlabeled [Sar¹-Ile⁵-Ile⁸]-Ang II (Sigma) as a ligand. After incubation, 3 ml of ice-cold 10 mM sodium phosphate (pH 7.4) containing 120 mM sodium chloride was added, and the mixture was immediately filtered through Whatman GF/F glass fibre filters (Whatman, Clifton, NJ) presoaked with 2 g/L human serum albumin. The filters were washed twice with 3 ml of the same buffer and counted in a gamma counter. The specific binding was calculated by subtracting the nonspecific binding, measured in the presence of 1 µM unlabeled [Sar¹-Ile⁵-Ile⁸]-Ang II, from the total binding. The dissociation constant (K_d) and the maximal binding capacity of ligand (B_max) were determined by Scatchard analysis of data obtained with unlabeled [Sar¹-Ile⁵-Ile⁸]-Ang II by using the RADLIG program for the analysis of radioligand binding experiments (Version 4; Biosoft, Cambridge, UK). The binding data were best fitted with a one-site fit. The Ang II receptor density was calculated by using the B_max and the protein concentration, assuming binding of one molecule of ligand to each receptor. In some cell membrane fractions, the Ang II receptor density was calculated by using the bound/free value, assuming binding characteristics (K_d) similar to those previously determined.

Measurement of Protein Concentration

The protein concentration in cell membrane fractions was determined by the method of Lowry et al. [17].

Measurement and Identification of Renin

Renin concentrations were measured by using the antibody-trapping method [18]. This method was previously described in more detail for the measurement of renin in bovine plasma, ovarian follicular fluid, and tissues [19–21]. The assay was standardized against a hog renin standard (Medical Research Council, Holly Hill, London, UK), and the renin concentrations are given as Goldblatt units (GU) per liter. Renin was identified as renin by inhibition of its enzymatic activity with 1 µM of the specific renin inhibitor remikiren (RO 42-5892; Hoffman-LaRoche, Basel, Switzerland).

Autoradiography

For in situ autoradiographic studies, the tissue was serial-sectioned (10 µm) in a cryostat at -16°C, and thaw-mounted on Polysine microscope slides (Menzel-Gläser, Braunschweig, Germany). The tissue sections were dried overnight at -2°C and used immediately or stored at -20°C until later use.

An in situ autoradiographic technique modified from Mendelsohn et al. [22] was used. Before assay, the sections were brought to room temperature and preincubated for 15 min in a buffer containing 10 mM sodium phosphate (pH 7.4), 120 mM sodium chloride, 5 mM MgCl₂, 1 mM EGTA (Sigma), 0.3 mM bacitracin (Sigma), and 2 g/L proteinase-free BSA (Biofac A/S, Copenhagen, Denmark). The sections were subsequently incubated in a humidified chamber for 180 min in a fresh identical buffer containing 0.5 nM ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II, a concentration close to the K_d value. Nonspecific binding was determined on serial sections using identical incubation conditions except for the addition of 1 µM of unlabeled [Sar¹-Ile⁵-Ile⁸]-Ang II. For localization and characterization of the Ang II receptor types, serial sections were incubated in the same buffer containing 0.5 nM ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II in the presence of 5 µM of losartan, in the presence of 5 µM of PD 123319, or in the presence of both. After incubation, the sections were rinsed four times (3 min each) in ice-cold 50 mM Tris-HCl (pH 7.4) and then dipped in ice-cold distilled water. A stream of warm air (60°C) was used to dry the sections quickly. The sections were fixed in paraformaldehyde vapors for 2 h at 80°C and allowed to evaporate overnight. Thereafter, they were dipped in photographic emulsion Ilford K2 (Ilford, Brønshøj, Denmark) diluted 1:2 in distilled water and exposed for 3 days at 4°C. The developing process with Kodak D-19 developer (Kodak, Farum, Denmark) was stopped with Liquid Acid Hardener (Ilford), and the sections were fixed in Rapid Fixer (Ilford) and washed for 1 h in rinsing water. After drying, the sections were weakly counterstained with hematoxylin and eosin in order to assist the localization of the silver grains.

The Ang II receptor density was estimated by a visual subjective scoring of the deposition of silver grains in the autoradiograms. The scoring was 0 (background), +, ++, and +++.

Statistical Analysis

The data were analyzed by using the Stat-100 statistical analysis package (Version 1.24; Biosoft). Analysis of skewness and kurtosis indicated that the data were most correctly analyzed by nonparametric statistics [23]. Values are given as median, with range in parentheses. A p value less than 0.05 was considered significant.

	RESULTS

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Characterization of Angiotensin II Receptors

Specific binding of ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II was found in all cell membrane fractions of placenta and fetal membranes from all 23 animals examined. The nonspecific binding was low and did not change during incubation for 5 h (n = 4). The time course of the specific binding in the placenta and fetal membranes indicated that equilibrium was reached after 45–60 min (n = 4). Accordingly, incubation for 60 min at 37°C was used in order to obtain equilibrium.

The specific binding of ligand in cell membrane fractions from placenta and fetal membranes approached saturation in experiments with ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II and increasing concentrations of unlabeled [Sar¹-Ile⁵-Ile⁸]-Ang II (Fig. 2a). The dissociation constant (K_d) determined by Scatchard plot (Fig. 2b) was 0.88 (0.32-1.51; n = 18) nM in the placentome. This value did not differ from those obtained in the intercotyledonary allantochorionic membrane (0.80 nM, range 0.40–1.24 nM; n = 18) and the allantoamnionic membrane (1.07 nM, range 0.27–1.56 nM; n = 11).

View larger version (23K): [in this window] [in a new window]   FIG. 2. a) Specific binding of ligand in a cell membrane fraction, prepared from bovine placentome in the second part of gestation, obtained by using 125I-[Sar1-Ile5-Ile8]-Ang II and increasing concentrations of unlabeled [Sar1-Ile5-Ile8]-Ang II. b) Scatchard analysis of the same data. In this experiment, the Kd and Bmax were 0.82 nM and 81 pM, respectively.

The proportions of the Ang II receptor types AT₁ and AT₂ were determined by displacement of the specific binding of ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II by the nonpeptide Ang II receptor antagonists losartan (DuP 753) and PD 123319 as shown in Figure 3. Losartan and PD 123319 are considered selective for the AT₁ and AT₂ receptors, respectively. Concentrations of losartan from 10^-6 to 10^-5 M fully displace ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II from the AT₁ receptor, whereas binding to the AT₂ receptor is only slightly affected [5, 6]. At higher concentrations, ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II is also displaced from the AT₂ receptor. Concentrations of PD 123319 from 10^-6 to 10^-5 M fully displace ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II from the AT₂ receptor and very slightly affect the binding to the AT₁ receptor [5, 6]. Accordingly, the proportions of the AT₁ and AT₂ receptors of the total Ang II receptors can be estimated in Figure 3 by the displacement by 10^-6 to 10^-5 M of losartan and PD 123319, respectively. The proportions of AT₂ receptors in total Ang II receptors in the placentome are shown in Table 1. These proportions did not change during gestation. In the intercotyledonary allantochorionic membrane (n = 12 animals from all 3 periods of gestation) and the allantoamnionic membrane (n = 12 animals from all 3 periods of gestation), the AT₂ receptors accounted for 80% to more than 90% of the Ang II receptors. No non-AT₁/non-AT₂ binding sites were detected by the Ang II receptor displacement curves.

View larger version (22K): [in this window] [in a new window]   FIG. 3. Representative displacement curves of the specific binding of 125I-[Sar1-Ile5-Ile8]-Ang II by unlabeled [Sar1-Ile5-Ile8]-Ang II (squares), PD 123319 (triangles), and losartan (circles) in a cell membrane fraction prepared from bovine placentome in the second part of gestation.

Angiotensin II Receptor Densities

The amount of Ang II receptor, related to the protein in cell membrane fractions from placentae and fetal membranes of various stages of gestation, is given as fmol/mg membrane protein and is shown in Table 2. The amount was lower in the placentome than in the intercotyledonary allantochorionic membrane (p < 0.001) and the allantoamnionic membrane (p < 0.001). It changed during gestation in the placentome (p < 0.005) and the intercotyledonary membrane (p < 0.02), but it did not change in the allantoamnionic membrane. The lowest values were found in the third part of gestation in the placentome and intercotyledonary membrane. The amount of Ang II receptor related to the wet tissue weight (fmol/g wet tissue) was calculated by using the same experimental data as in Table 2 and is given in Table 3. Analyzed in this way, the values were lower in the allantoamnionic membrane than in the placentome (p < 0.01) and intercotyledonary membrane (p < 0.005). It changed in the placentome (p < 0.005) and was highest in the second part of gestation. No changes were found in the intercotyledonary membrane and allantoamnionic membrane.

Renin Concentrations

The renin concentrations in bovine placentae and fetal membranes (Table 4) were low or, in several animals, even below the detection limit (0.014 GU/kg wet tissue weight). In the intercotyledonary membrane and the allantoamnionic membrane, no renin could be detected during the first part of gestation. All measured renin was completely inhibited by the specific renin inhibitor remikiren, indicating that no nonrenin enzymes, capable of forming Ang I, were present in the placenta and fetal membranes.

Autoradiography

In the intercotyledonary allantochorionic membrane, most of the ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II binding occurred in the mesenchymal tissue. It was localized to mesenchymal cells adjacent to the trophoblast cell layer and around the larger and smaller arteries, and, with less intense staining, in close relationship to the allantoic endoderm (Fig. 4). In accordance with the Ang II receptor binding study, the predominant Ang II receptor type was the AT₂ receptor (Fig. 4a). The less pronounced AT₁ receptor binding appeared in the same localizations as the AT₂ receptors (Fig. 4b). The allantoic endoderm and the trophoblast cells did not bind ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II. The uterine epithelium did not bind ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II, whereas the lamina propria of the endometrium mainly revealed AT₁ receptor binding (Fig. 4b), less AT₂ receptor binding (Fig. 4a), and only scarce non-AT₁/non-AT₂ Ang II binding sites (Figs. 4e and 5b).

View larger version (163K): [in this window] [in a new window]   FIG. 4. Autoradiograms showing the distribution of 125I-[Sar1-Ile5-Ile8]-Ang II binding in serial cryosections of a smooth part of the bovine placenta (Day 145). Dark regions indicate high density of labeled receptors. a) Binding in the presence of losartan showing AT2 receptor binding. b) Binding in the presence of PD 123319 showing AT1 receptor binding. c) Total binding. d) Binding in the presence of 1 µM unlabeled [Sar1-Ile5-Ile8]-Ang II. e) Binding in the presence of both PD 123319 and losartan. *, Artery; MES, fetal (somatopleural) mesenchyme; arrowhead, trophoblast layer; open arrow, uterine epithelium; LP, lamina propria of the endometrium. Bar = 150 µm.

View larger version (81K): [in this window] [in a new window]   FIG. 5. Autoradiograms showing the distribution of 125I-[Sar1-Ile5-Ile8]-Ang II binding in serial cryosections of a smooth part of the bovine placenta (Day 115). Black silver grains indicate presence of labeled receptors. a) Total binding. b) Binding in the presence of both PD 123319 and losartan. c) Binding in the presence of 1 µM unlabeled [Sar1-Ile5-Ile8]-Ang II. MES, Fetal mesenchyme; solid arrows, trophoblast layer; open arrows, uterine epithelium; LP, lamina propria of the endometrium. Bar = 60 µm.

In the allantochorionic membrane of the placentomes (cotyledon), the AT₂ receptor was also the most abundant Ang II receptor type (Figs. 6a and 7c). Most binding occurred on the mesenchymal cells at the fetal side of the cotyledon and at the lower-order fetal villi (Fig. 6a). This binding disappeared in the higher-order villi. Specific binding to the tunica media of the arteries was observed in only a few arteries and was always scarce. Again, no Ang II receptor expression was observed on the allantoic endoderm and trophoblast cells. No binding of ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II was demonstrated in relation to the bovine placental lactogen-producing binucleate and trinucleate cells in the trophoblast cell layer and maternal uterine epithelium (Fig. 8). On the maternal side of the placentome (caruncle), the lamina propria of the endometrium and the lower-order septa of the maternal crypts showed binding (Fig. 6b). In contrast to the fetal compartment, the main Ang II receptor type in the maternal constituent was the AT₁ receptor (Figs. 6b and 7d). No binding was found on the higher-order septa of the maternal crypts. The binding in the mesenchymal tissue revealed a characteristic patchy pattern (Fig. 9), as most silver grains accumulated in close relationship to nuclei of mesenchymal cells. Apparently, the mesenchymal cells differed with respect to Ang II receptor expression, since cells within the same area showed marked differences in Ang II binding (Fig. 9).

View larger version (130K): [in this window] [in a new window]   FIG. 6. Autoradiograms showing the distribution of 125I-[Sar1-Ile5-Ile8]-Ang II binding in serial cryosections of a bovine placentome (Day 145). Dark regions indicate high density of labeled receptors. a) Binding in the presence of losartan showing AT2 receptors. b) Binding in the presence of PD 123319 showing AT1 receptors. c) Total binding. d) Binding in the presence of 1 µM unlabeled [Sar1-Ile5-Ile8]-Ang II. F, Fetal side; solid arrows, mesenchyme of fetal villi; M, maternal side; open arrows, maternal crypt wall. Bar = 1000 µm.

View larger version (175K): [in this window] [in a new window]   FIG. 7. Detail of serial cryosections of a bovine placentome (Day 152). a) Brightfield view of a hematoxylin and eosin-stained section. b–f) Brightfield photomicrographs combined with epipolarised light of emulsion-dipped sections. Bright grains indicate the presence of 125I-[Sar1-Ile5-Ile8]-Ang II-labeled receptor. b) Total binding. c) Binding in the presence of losartan, demonstrating AT2 receptors. d) Binding in the presence of PD 123319, demonstrating AT1 receptors. e) Binding in the presence of both PD 123319 and losartan, demonstrating non-AT1/non-AT2 binding sites. f) Binding in the presence of 1 µM unlabeled [Sar1-Ile5-Ile8]-Ang II. M, maternal crypt; F, fetal villus; solid arrows, trophoblast cells covering the fetal villi; open arrows, uterine epithelium covering the maternal crypt. Bar = 75 µm.

View larger version (146K): [in this window] [in a new window]   FIG. 8. Brightfield photomicrograph combined with epipolarised light showing a detail of emulsion-dipped cryosection of a bovine placentome (Day 235). Bright grains indicate the presence of 125I-[Sar1-Ile5-Ile8]-Ang II-labeled receptors. F, Fetal villus; M, maternal crypt; solid arrows, trophoblast cells covering the fetal villi; open arrows, uterine epithelium covering the maternal crypt; white asterisks, binucleate (migrating) cells. Bar = 75 µm.

View larger version (158K): [in this window] [in a new window]   FIG. 9. Detail of a bovine placentome (Day 235). Brightfield photomicrograph of a hematoxylin and eosin-stained, emulsion-dipped section showing 125I-[Sar1-Ile5-Ile8]-Ang II binding to AT2 receptors of fetal mesenchymal cells in the presence of losartan. Black silver grains indicate presence of labeled receptors. Bar = 35 µm.

The ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II binding in the allantoamnionic membrane was always relatively weak (data not shown). It occurred mainly in the mesenchyme in close relation to the allantoic endoderm and amnionic ectoderm, and around the few arteries. The predominant Ang II receptor type was the AT₂ receptor. The allantoic endoderm and amnionic ectoderm did not reveal any binding.

Displacement studies using both losartan and PD 123319 revealed non-AT₁/non-AT₂ Ang II binding sites mainly located in the placentome, namely in the lamina propria of the endometrium and on the lower-order septa of the maternal crypts. In all fetal compartments, only slight binding was observed. In three out of five allantochorionic intercotyledonary membranes and in three out of three allantoamnionic membranes, most of the non-AT₁/non-AT₂ Ang II binding sites were located in the mesenchymal tissue.

No difference in the pattern of distribution of Ang II receptors was observed throughout gestation.

	DISCUSSION

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The bovine placenta and fetal membranes contained high densities of Ang II receptors in all three stages of gestation. By using type-specific nonpeptide Ang II antagonists, Ang II receptor binding studies as well as autoradiography showed that these Ang II receptors were predominantly AT₂ receptors. This is in accordance with recent findings in the pig [14] but differs from findings in the human placenta, in which most Ang II receptors were AT₁ receptors [15]. The bovine and the porcine placenta are both epitheliochorial, with the bovine of the subtype synepitheliochorial, whereas the human placenta is hemomonochorial [24]. It is very likely that the type of Ang II receptor that predominates in the placenta relates to the differences in placental architecture.

Autoradiography indicated the presence of non-AT₁/non-AT₂ Ang II binding sites at very low densities in the bovine fetal membranes as well as in the placentome. By a visual estimate, these binding sites represented 1% or less of the total amount of Ang II receptors, and they were undetectable in the Ang II receptor binding studies. AT₁ and AT₂ receptor antagonist concentrations of 5 µM, which are much higher than their IC₅₀ values [5, 6], were used in order to displace the ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II binding. Although both losartan and PD 123319 were used together at these high concentrations, specific binding was observed in the lower-order septa of the maternal crypts and in the fetal membranes. Non-AT₁/non-AT₂ Ang II binding sites have previously been reported in the human endometrium [7], but to the knowledge of the authors, no previous reports of non-AT₁/non-AT₂ Ang II binding sites in the fetal membranes have been made.

Autoradiography showed a marked ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II binding in the intercotyledonary membrane and the placentome, whereas the binding to the allantoamnionic membrane appeared less intense. Data from Ang II receptor binding studies showed, however, that the amount of receptor related to the extracted cell membrane protein (Table 2) was lower in the placentome than in the allantoamnionic and intercotyledonary membrane. The allantoamnionic membrane is a tissue that contains relatively few scattered cells, whereas the intercotyledonary membrane and the placentome have a much higher cell density. In these tissues, much more cell membrane protein can thus be extracted per gram of tissue. Therefore, the amount of Ang II receptor per gram wet tissue was higher in the placentome than in the allantoamnionic membrane (Table 3). This corresponds with the Ang II receptor densities observed in the autoradiograms. Our findings underline the possibility that receptor densities obtained by visual subjective scoring or by computer-assisted conversion of the optical density of autoradiograms, as in reference [25], may not be comparable with those obtained by receptor binding studies, in which the amount of receptor is related to the cell membrane protein as in Table 2 in this paper.

In the human syncytiotrophoblast, the AT₁ receptors were localized on the basolateral plasma membranes [26]. Recent in vitro studies using human trophoblast cells have demonstrated that Ang II stimulated human placental lactogen and specific ß1-glycoprotein secretion through the AT₁ receptor [12]. As shown in Figure 8, we found no binding of ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II to the bovine placental lactogen-producing binucleate and trinucleate cells situated in the trophoblast cell layer and maternal uterine epithelium [27]. This finding suggests that Ang II does not regulate the secretion of placental lactogen in the bovine placenta, in contrast to the findings in the humans [12].

In the bovine species, the Ang II receptors were localized to cells in the mesenchyme of the fetal membranes and were mainly AT₂ receptors. The fact that the AT₂ receptor predominated in the fetal compartment of the bovine placenta and the finding that the density of the Ang II receptor decreased at the end of the gestation suggest that the function of RAS in the bovine placenta differs from that in humans. In the rodent and primate fetus, the presence of abundant Ang II binding sites has been found in locations at which Ang II binding is not observed in the adult [28, 29]. In contrast to the classical Ang II target tissues, which contain mostly AT₁ receptors, the majority of the fetal Ang II binding sites are AT₂ receptors. They are located in the fetal skin, skeletal muscle, and mesenchymal cells and disappear after birth [29]. On the basis of the location and ontogenicity of the expression of the AT₂ receptors, it was suggested that they may be involved in cell differentiation and growth. Recent experiments using vascular tissue cultures showed that the AT₂ receptors exert an antiproliferative effect by counteracting the AT₁ receptor growth-promoting effect [30]. We found AT₁ and AT₂ receptors at the same locations in the fetal and maternal tissue, although in different densities. This could indicate that Ang II exerts its functions through one receptor type (AT₁) and modulates them through the other type (AT₂). Further studies, however, are needed to clarify whether both receptor types are located on the same mesenchymal cells or on different cells within the same area of the fetal membranes. Non-AT₁/non-AT₂ Ang II binding sites were found at low densities in the bovine fetal membranes and in the placentome and the lamina propria of the endometrium. These bindings sites might also regulate growth, since Ang II was shown to stimulate proliferation of human keratinocytes in primary culture via non-AT₁/non-AT₂ Ang II binding sites [31].

In the placentomes, we found a predominance of AT₂ receptors. The highest proportion of AT₁ receptors, determined by Ang II receptor binding studies, was found at the beginning of the gestation. However, autoradiography showed that the AT₁ receptors were placed mostly on the maternal side, whereas the AT₂ receptors predominated on the fetal side. In the early placentome, no stalk has developed to indicate a natural border towards the endometrium. Our finding of a higher proportion of AT₁ receptors in the first part of gestation probably reflects a higher content of maternal tissue in the early placentomes.

Previous studies showed that Ang II stimulated angiogenesis [32] and regulated uteroplacental blood-flow [8–10]. Autoradiography showed very little ¹²⁵I-[Sar¹-Ile⁵-Ile⁸]-Ang II binding in either arteries or minor blood vessels in the bovine placenta. Ang II, however, may still exert such effects in the vasculature of the bovine placenta, since a low density of Ang II receptors does not exclude an even marked effect of Ang II.

Active renin concentrations in the bovine placenta were either undetectable or low throughout the gestation. This is in accordance with previous findings [20]. In the present study, enzymatically inactive renin (prorenin) was not determined. In an earlier study, however, no prorenin was detected in the bovine placenta [20]. This is in contrast to the human placenta, which—with the kidney—is a main source of the increased prorenin in the plasma during pregnancy [33, 34]. The active renin concentration is the rate-limiting factor for the formation of Ang I, which is rapidly converted to Ang II by the ubiquitous angiotensin-converting enzyme. The finding that active renin was very low or undetectable in the bovine placenta indicates a low formation rate of Ang II in this tissue. Other enzymes, however, are capable of forming angiotensin from angiotensinogen [35]. In the present study, the specific renin inhibitor remikiren abolished all formation of Ang I in the renin assay, indicating that no other Ang I-forming enzymes are present in the bovine placenta.

In conclusion, all parts of the bovine placenta and fetal membranes contained high densities of Ang II receptors. AT₂ receptors were highly expressed in the fetal part of the placenta, whereas AT₁ receptors predominated in the maternal part. No changes in the pattern of distribution of the Ang II receptors were found throughout gestation. It is suggested that Ang II exerts an effect on regulatory as well as growth processes in these tissues.

	ACKNOWLEDGMENTS

 
The skillful technical assistance of Susanne Holm and Iben Thomsen is gratefully acknowledged. The renin inhibitor remikiren (RO 42-5892) was kindly provided by Dr. Walter Fischli, Hoffman-LaRoche, Basel, Switzerland. The nonpeptide type-specific Ang II receptor antagonists losartan (DuP 753) and PD 123319 were gifts from the Du Pont Merck Pharmaceutical Company, Wilmington, DE, and Parke-Davis, Ann Arbor, MI, respectively.

	FOOTNOTES

 
¹ This study was partially supported by grants from the Danish Agricultural and Veterinary Research Council, the Nordic Insulin Fund, and the NOVO Fund.

² Correspondence: Kirsten H. Schauser, Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, 13 Bülowsvej, DK-1870 Frederiksberg C, Denmark. FAX: 45-35282525; kirsten.h.schauser{at}iaf.kvl.dk

Accepted: April 30, 1998.

Received: July 7, 1997.

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