Angiotensin ii receptor in human placental syncytiotrophoblast plasma membranes

Life Sciences, Vol. 58, No. 11, pp. 8?7-882,19% rzopyright Q 1996 Ekmier science Inc. Printed in the USA. All righk reserved cm24-m/!36 $15.00 t 40

PII SOO24-3205(96)00030-6

ANGIOTENSIN II RECEPTOR SYNCYTIOTROPHOBLAST

IN HUMAN PLACENTAL PLASMA MEMBRANES

Eugenio Jimenez, Manuel Murioz, Jose Pavia* and Mercedes Montiel Department of Biochemistry and Molecular Biology and Department of Pharmacology* Faculty of Medicine. University of Malaga. 29080-Malaga, Spain. (Received in final form January 3, 1996)

Summary

Previous studies have reported the presence of high-affinity angiotensin II (Ang II) binding sites in human placental tissue, but it has not been determined whether these are located in brush border (BBM) or basolateral plasma (BPM) membranes of the syncytiotrophoblast. Our findings provide no evidence for Ang II receptors in BBM, yet they reveal a single class of binding sites in BPM preparations (Kd of 4.08iO.61 nM and B_ of 2368.7658.2 fmol/mg protein). Pharmacological characterization also revealed that this receptor was an AT1 receptor subtype. Moreover, isoelectric focusing analysis demonstrated a predominant Ang II-receptor complex migrating to pl 7.0, and two minor receptors at pl 7.2 and 6.5. These data suggest a physiological role of the renin-angiotensin system on syncytiotrophoblast BPM in regulating placental function. Key Words: angiotensin II receptor isoforms, syncytiotrophoblast

plasma membranes

Angiotensin II (Ang II) is a biologically active peptide of the renin-angiotensin system (RAS) which exerts a variety of physiological effects in the cardiovascular, endocrine and nervous system through the stimulation of specific receptors. A number of studies have provided evidence for the presence of the RAS components in the uteroplacental organ, such as renin substrate, renin, angiotensin converting enzyme and angiotensins (1). Moreover, several studies have described the existence of specific Ang II binding sites in human placenta (2-4). These observations strongly suggest a local role of the RAS in the regulation of the placental function. Although two classes of Ang II binding sites were initially described in plasma membranes from human term placenta (2), recent data indicate the presence of a single class of binding sites (3). Moreover, further characterization of these binding sites using selective Ang II receptor subtype ligand losartan (AT1 receptor blocker) and CGP42112A (AT2 receptor blocker) showed that only the AT1 receptor subtype --____---Corresponding Author: Eugenio Jimenez, Department of Biochemistry and Molecular Biology, Faculty of Medicine. University of Malaga, 29080_Malaga, Spain.

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was present in human placental tissues (4). The syncytiotrophoblast cell constitutes the main barrier to maternal-fetal transport and exchange of minerals and nutrients between the mother and the fetus (5). Since syncytiotrophoblast basolateral plasma membranes (BPM; facing the fetus) and brushborder plasma membranes (BBM; facing the mother) exhibit different functions and properties (6) the present study was designed to localize the Ang II receptors in human syncytiotrophoblast plasma membranes, and to obtain more information on their molecular structure. Material and Methods Preparation and characterization of syncytlotrophoblast plasma membranes: Human term placentas were obtained within 30 min after normal vaginal delivery and placed in ice-cold NaCl 0.9%. All the procedures were carried out at 4°C. The umbilical cord, amnion, chorion and fibrous membranes were removed and the syncytiotrophoblastic tissue was cut into small pieces with scissors. BBM were prepared using a homogenization and Mg2+ precipitation technique (7) and BPM were obtained according to the method described by Kelley et al. (8). Both syncytiotrophoblast BPM and BBM preparations were resuspended in 5 mM HEPESTris, pH 7.4, containing proteinase inhibitors (1 pg/ml leupeptin, 1 &ml aprotinin, 0.1 @/ml bacitracin and 0.1 pg/ml phenylmethanesulphonyl fluoride), and were frozen and stored at -80°C until use for Ang II binding assays. The purity of preparations were assessed by measuring the enrichment of the brush-border enzyme alkaline phosphatase (9) and the basal plasma marker, sH-dihydroalprenolol binding (8). 125/-Ang /I binding assay: Binding of ‘25l-Ang II (specific activity 2200 Ci/mmol; DuPont-New England Nuclear) to BPM and BBM samples were conducted in 150 PI of 50 mM Tris-HCI, pH 7.4, containing 120 mM NaCI, 6 mM MgCl2, 0.1% (w/v) bovine serum albumin (BSA) and proteinase inhibitors. Samples (50 pg protein) were incubated at room temperature (22°C) for 45 min with 0.1 nM 12sl-Ang II and varying concentrations of unlabeled Ang II (Sigma Chemical Co.) or selective Ang II receptor subtype ligands (losartan and CGP42112A). Bound and free radioactivity were separated by adding 3.0 ml of ice-cold saline to the assay tube followed by vacuum filtration. The assay tube and filter were rinsed with three additional washes of 3.0 ml of cold saline and then the filters were counted in a gamma-counter. Equilibrium dissociation constant (&) and concentration of receptor sites (Bmax) were determined using the iterative curve-fitting program “Ligand”. Nonspecific binding was determined in the presence of 1 PM unlabeled Ang II, and substrated for each experiment in order to calculate the specific binding. lsoelectrlc focusing analysis: In isoelectric focusing (IEF) studies, BPM samples (50 pg protein) were incubated in the same conditions above indicated. Bound and free 125l-Ang II were separated by centrifugation at 10000 g for 5 min. The pellet was resuspended and washed once more with assay buffer and recentrifuged. The resultant pellet was resuspended in 100 ~1of Tris buffer, containing 0.5% Triton X-100, incubated for 20 min at 4°C and centrifuged at 10000 g for 5 min. The solubilized membrane proteins were collected and fractionated by IEF in polyacrylamide slab gels containing 12% glycerol. A pH 3.5-10.0 gradient was achieved with 0.7% ampholine pH 3510.0 and 3% ampholine pH 5.0-8.0 (LKB). Gels were prefocused for 40 min at 2000 V, 20 mA and 20 W, and the runs were carried out for 4 h at 2500 V, 20 mA and 20 W. Gels were then cut into 3 mm width slices and counted in a gamma-counter.

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Protein deferrWn8tiOff.’ Protein concentrations were determined according to Lowry et al. (lo), using BSA as standard. Results Membrane markers: The purity of preparations were assessed by measuring the enrichment of the brush-border enzyme alkaline phosphatase and the basal plasma marker sH-dihydroalprenolol binding. Alkaline phosphatase activity in the BBM was enriched on average 1Sfold compared to the homogenate. In contrast there was little contamination by alkaline phosphatase in BPM (enrichment factor 1-2-fold). sHdihydroalprenolol binding in BPM was more than 30-fold greater than binding in the homogenate (Table 1). TABLE 1 Parameters of syncytiotrophoblast membranes in BPM and BBM of normal term human placenta. Membrane

Homogenate BBM BPM

Alkaline phophatase (~mol/min/mg protein)

sH-dihydroalprenolol binding (fmol/mg protein)

l.OW.18 20.45k4.80 2.1 l&O.90

90.5k8.1 87.4k7.2 2843.1k313.8

Values are mean*SD of 5 separate experiments Binding assays: Scatchard analysis from saturation experiments indicated a single population of Ang II binding sites in human placental syncytiotrophoblastic BPM (Kd of 4.08kO.61 nM and B,, of 2368.7658.2 fmol/mg protein) (Fig. 1). There was no specific binding of Ang II in BBM preparations.

Bound (PM)

11

10

9 8 Ang II (- log M)

7

6

FIG. 1 Competitive binding of 12%Ang II to human placental syncytiotrophoblast BPM (0) in the presence of the designated concentrations of unlabeled Ang II. Scatchard analysis of one determination in BPM preparation is shown in the inset.

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Monophasic displacement of 1251-Ang II in the presence of the non-peptide AT, receptor ligand losartan or the AT2 receptor ligand CGP42112A are shown in Fig 2. Losartan was capable of competing for these binding sites (4 of 208551 nM), while no changes were observed in the presence of lower concentrations of CGP42112A. 100

Ligsnd

(-

log

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FIG. 2 Competitive binding of 1251-Ang II to human placental syncytiotrophoblast BPM in the presence of the designated concentrations of unlabeled Ang II (a), losartan (0) or CGP42112A (I). Isoelectric focusing assays: A representative pattern from IEF analysis of the 125lAng II-receptor complex isoforms in syncytiotrophoblastic BPM preparations is illustrated in Fig. 3A. Three 125l-Ang II-binding complexes were observed at pl 7.2, 7.0 and 6.5, representing 8. li1.9%, 79.9*3.1% and 12.0*1.3% (mean&D, n=3) of the specific 125l-Ang II bound, respectively. Moreover, an additional small spot of radioactivity at pl 6.7 was noted in the presence of unlabeled Ang II, which may represent a non-specific ‘25l-Ang II binding site. Losartan, a selective AT1 subtype ligand, inhibited 125l-Ang II binding at these three specific isoforms, while no changes were observed in the presence of the selective AT2 subtype ligand CGP 42112A (Fig. 38).

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FIG. 3 IEF profile of 125l-Ang II labeled receptors from human placental syncytiotrophoblast BPM: (A) in the presence (0) or absence (0) of unlabeled Ang II, or (B) in the presence of 1 KM losartan (A) or 0.5 PM CGP42112A (a).

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It has been difficult to interpret and draw conclusions on the characterization of Ang II receptor from previous studies using whole human placenta. In the present study, Ang II receptor localization in human sync~otr~~blast plasma membranes was investi~t~. Obviously, the purity of the membrane is the first irn~~nt step for the interpretation of the binding data. Our values of enrichment suggest a high degree of purification of BBM and BPM preparations, and in accordance with these previous findings, binding data indicate the presence of specific receptors for Ang II in human placental syncytiotrophoblast BPM. There is no evidence for any Ang II receptors in BBM. ‘2sl-Ang II labeled a single population of binding sites in BPM pr~arations with a Kd and Bmax in good agreement with previously reported data (2-4). Two distinct Ang II receptor subtypes (AT1 and AT2) have been identified (1 l-l 2). Binding-inhibition studies, using selective Ang II receptor subtype ligand losartan (AT1 receptor blocker) and CGP42112A (AT2 receptor blocker), revealed the presence of only one type of Ang II binding sites in these sy~~iotrop~blast BPM, ~rre~o~ing to the AT1 receptor subtype. Recently, two closely related AT1 forms (AT1 A and AT18) encoded by different genes have been identified in the rat and human (13-l 4). AT1 8 subtype differs pharmacologically from ATlA showing PM as opposed to nM Ki for losartan (15-16). Accordingly, our & data for losartan suggests that the AT1 B form could be predominantly expressed in syncytiotrophoblast BPM. Recently, Ang II has been shown to stimulate estradiol secretion from human placental explants in a dose- and time-dependent manner (17). Nevertheless, other physiological and biochemical functions (i.e. fluid and ion transport between the mother and the fetus, fetal volume homeostasis or regulation of vascular permeability) associated with this specific binding of Ang II to placental membrane remain to be investigate (1). Interestingly, our observations demonstrati~ a polar l~lization of Ang II binding sites in human sync~iotrophobiast tissue support the hypothesis that Ang II produced by the fetus have a direct regulatory role on the BPM; in addition, our finding point to Ang II binding as a new syncytiotrophoblast BPM marker. The molecular structure of the placental Ang II receptors using phot~ffini~ labeling ex~riments has been performed (3) sowing that the ligand-binding unit complex migrated as a single specific band with an apparent molecular weight of 92 kDa (3). In order to obtain more details on the molecular structure of the Ang II receptor in human syncytiotrophoblast cell solubilized 125l-Ang II receptor-complexes from BPM preparations were analyzed by IEF on polyacrylamide gels. Our data demonstrated three 125l-Ang II receptor-complexes, a pre~minant isoform migrating to pl 7.0, and two minor migrating to pl 7.2 and 6.5. Under the same experimental conditions the existence of a tissue-dependent Ang II receptor isoform profiles in rats have been found (18-20). The effect of different antagonists on adrenal gland Ang II receptor isoforms shows that the pl 7.0 and 6.8 isoforms exhibit predominantly AT1 pharmacology, and identifies the pl 6.3 isoform as predominantly AT2 receptor (18-19). It therefore appears that the molecular structural properties of Ang II receptors may differ depending on species and/or tissues. These differences could be related to the individual effects exerted by Ang II upon its several target tissues and/or the processing which accompanies receptor internalization, desensitization or recycling. Further investigation is necessary to identify the precise nature and function of these Ang II receptor isoforms.

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Acknowledrrements We would like to thank Dr. Oliva (Servicio de Ginecoiogia y Obstetricia, Hospital Clinic0 Universitario) for providing the human placentas. We would also like to thank Dr. Marc de Gasparo of CIBA-GEIGY Limited (Basel, Switzerland) for providing CGP42112A and losartan. M. Mufioz gratefully acknowledges Prof. D.L. Yudilevich for his advice in isolating placental membranes. This work was supported by a grant PB91-0764 from the Direction General de lnvestigacibn Cientifica y Tecnica. Spain.

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