Characterization of angiotensin II receptors in the rat fetus

Peptides, Vol. 10, pp. 459--463. PergamonPress plc, 1989. Printed in the U.S.A.

0196-9781/89 $3.00 + .00

Characterization of Angiotensin II Receptors in the Rat Fetus C A R L A J O N E S , l M O N I C A A. M I L L A N , F R E D E R I C K N A F T O L I N l AND GRETI AGUILERA 2

Section on Endocrine Physiology, Endocrinology and Reproduction Research Branch National Institute of Child Health and Human Development, Bethesda, MD 20892 R e c e i v e d 13 O c t o b e r 1988

JONES, C., M. A. MILLAN, F. NAF'rOLIN AND G. AGUILERA. Characterization of angiotensin H receptors in the rat fetus. PEPTIDES 111(2) 459-463, 1989.--The presence of All receptors during early and late embryonic development was studied by binding of 125I[Sar t ,IleS]AII to whole mouse blastocysts and membrane-rich fractions from rat conceptuses, 7 to 21 days in gestation. In early mouse embryos there was no detectable binding under a variety of experimental conditions. However, in late gestation rat fetuses, specific and high affinity binding was observed, with a concentration of sites similar in membranes from whole and eviscerated fetuses. Using less than 100 ixg of membrane protein, binding was time and temperature dependent, maintaining equilibrium from 30 to 120 min at 23°C and it was enhanced by addition of Mg +2 up to 5 mM, EGTA 2 mM and dithiothreitol up to 2.5 mM. Scatchard analysis of the binding data indicated Kd values ranging between 0.7 and 0.9 nM. Binding was first detectable at day 10 (14.3 ---2.3 fmol/mg), increasing to 104 ± 16, 2,625 ___168, 5,993 --_ 152 and 5,902 ~ 92 by days 12, 15, 18, and 21 of gestational age, respectively. Since the functional significance of these binding sites depends on the availability of the agonist ligand, acid extracts from eviscerated 10-day-old fetuses were analyzed for the presence of All. Measurement of All by radioimmunoassay revealed immunoreactive AII-like material (845 pg/g of tissue), with an elution pattern identical to that of AII standard in a Sephadex G-50 column. This material was bioactive, as demonstrated by its ability to displace J25I[SarJ,llea]AII from adrenal glomerulosa membranes, an effect which was abolished by pretreatment of the extract with All antibody. The overall data, particularly the high concentration of AII binding with characteristics similar to All receptors in recognized target tissues in both visceral and nonvisceral sites in the fetus, suggest that the octapeptide may have an important role during fetal development. Angiotensin II receptors

Fetal development

Radioimmunoassay

METHOD

EVIDENCE is accumulating to suggest that the role of the renin-angiotensin system extends beyond its recognized effects in circulatory homeostasis (2, 10, 12, 14, 18, 21). Components of the system have been found in a number of tissues unrelated to the control of blood pressure and mineralocorticoid secretion, including the reproductive tract in humans and experimental animals (9, 12, 18, 21, 23, 31). All receptors, which are required for the peptide to have a biological effect, are present in ovary, testes, uterus, placenta, chorion and amnion (2, 8, 11, 17, 27, 34). Recent studies have shown that administration of an All antagonist to PMSG/hCG primed immature rats inhibits ovulation (26) and that treatment of preimplantation mouse embryos in vitro with All accelerates maturation (22). Because these studies suggest that All may have an important role in reproductive function, including embryonic development, we sought to identify receptors for All that may mediate the action of the peptide during fetal growth. The results show the presence of increasing number of AII receptors in the eviscerated fetus from days 12 to 21 of gestational age.

All, [Sar~,AlaS]AlI, [Sarl,IleS]AII, desAsp]-AII (AIII) and other peptides were purchased from Peninsula, Belmont, CA. t25I-labelled [Sar t ,IleS]AII was prepared by modified chloramineT radioiodination and purified by HPLC on a C-18 column using acetonitrile/0.1 M ammonium bicarbonate (15:85) buffer pH 8.0. The specific activity of the tracer ranged from 1,500 on 1,800 txCi/Ixg as determined by radioreceptor assay using rat adrenal capsular membranes. Maximum binding activity of the tracer to adrenal membranes was 50 to 70% (15). For early embryo studies, female mice were primed with PMSG/hCG and mated. Twenty-four hr later animals were killed by cervical dislocation and the oviduct with a small portion of uterine horn attached was removed and placed in a small volume of Ham's F-10 media containing penicillin and streptomycin. Fertilized eggs were flushed from the oviduct with a syringe attached to a 18 gauge needle. Healthy 2-cell embyros were

~Department of OB/GYN Yale University, New Haven, CT. 2Requests for reprints should be addressed to Greti Aguilera, M.D., Section on Endocrine Physiology, ERRB, NIH, Bldg. 10, Rm. 8C-407, Bethesda, MD 20892.

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FIG. 1. Binding of 125I[Sarl ,lleS]AII to membrane-rich fractions prepared from whole rat fetus or eviscerated fetal carcasses. Points are the mean of duplicate incubations in a representative experiment. selected under a dissecting microscope, removed by aspiration and cultured for 72 hr in the same media containing 1% BSA at a density of 10 embryos per dish. Embryos at the stage of a 60-70 cell blastocyst were selected for the binding studies. Groups of 25-100 embryos were pooled, incubated in culture media with J25I[Sar~,IleS]AII for different periods of time in the presence and in the absence of 1 ~M unlabelled All. For studies in fetuses, pregnant female Sprague-Dawley rats, between 7 and 21 days of gestation, were obtained from Zivic Miller Laboratories Inc. (Zelienople, PA). The day of gestation was estimated from the date of insemination. Pregnant rats were killed by decapitation, the embryos rapidly removed from the uterus, separated from the membranes, and placed in ice-cold phosphate buffered saline, Whole early embryos 7 to 12 days old and whole and eviscerated fetuses from 15 to 21 days gestation were homogenized in 10 volumes of ice-cold 20 mM NaHCO3 by 6 strokes of a mechanical homogenizer (Tekmar, Cincinnati, OH). The homogenate was stirred on ice for 20 rain, filtered through nylon gauze, and centrifuged at 1,000 and 30,000 × g. The pellet was resuspended to give a protein concentration of about 500 ixg/ml. Aliquots of protein suspension (30 to 70 txg) were incubated with 100,000 cpm ~25I[Sar~,IleS]AI1 (0.2 nM), in a total volume of 250 ~1 of 50 mM Tris HCI buffer pH 7.4 containing 2.5 mM MgC12, 2.0 mM EGTA, 2.5 mM DTT, 0.5% bovine serum albumin, and increasing concentrations of unlabelled All. After a 60 min incubation at 24°C, bound radioactivity was separated by filtration through GF/C glass fiber filters, washed twice with 4 ml phosphate buffered saline (PBS), and the radioactivity analyzed in a ",/spectrometer (15). Nonspecific binding determined in the presence of 1 I~M unlabelled AII was about 50% of the total bound radioactivity for early embryos and less than 10% for embryos 15 days of gestation and more. Affinity and concentration of the binding sites were calculated by computer analysis of the binding data (24), using the plots described by Berson and Yallow (4) and Rosenthal (29), commonly identified as Scatchard plots. To determine whether the octapeptide All is present in the fetus, eviscerated 19-day-old rat conceptuses were immediately homogenized in 10 volumes of 2 N acetic acid using a mechanical homogenizer (Tekmar, Cincinnati, OH), placed in a water bath at 100°C for 10 rain and centrifuged at 30,000 × g for 30 min. Prior to homogenization, radiodinated All (8,000 cpm) was added to measure recovery following extraction and separation procedures. The clear supernatant was recirculated 3 times through a SepPak C-18 cartridge, eluted with 60% acetonitrile in 0.1% TFA and

FIG. 2. Time course of the binding of 125[Sarl,IleS]AII to rat fetal membrane-rich fractions. Seventy I~g of membrane protein were incubated with 100,000 cpm radioligand (123 pM). During the plateau, bound radioactivity was 48% of the total added. Points are the mean of the specific binding values in duplicate incubations in 1 of 4 similar experiments.

lyophilized in a vacuum centrifuge. The residue was resuspended in 500 ILl of 50 mM acetate buffer pH 4.5, applied to a 40 × 1 cm Sephadex G-50 column, and eluted with the same buffer. AII content in the fractions was determined by RIA (7) using an antibody specific for the C-terminal part of the molecule which recognizes equally AII and AIII. To assess the bioactivity of the extracted material, fractions containing immunoreactive material were pooled, lyophilized, resuspended in PBS and aliquots incubated with AII antibody (1:500 final concentration) or normal rabbit serum. After overnight incubation at 4°C, bound material was precipitated with antirabbit serum and polyethylenglycol, and the supernatant extracted with a C-18 SepPak cartridge as described above. The lyophylized residue was dissolved in All binding buffer and tested

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FIG. 3. Specificity of AII binding in the rat fetus as shown by the ability of AII analogues to displace the binding of 12~I[Sar~,IleS]AIIfrom membrane rich fractions. Points are the mean of duplicate incubations in l of 3 similar experiments.

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FIG. 4. All binding to membranesprepared from rat fetuses at different gestational ages. Points are the mean of duplicate incubations in a representative experiment. for binding activity in a radioreceptor assay using adrenal glomerulosa membranes (15). Unless specified all data are expressed as the mean and SE of the values in several experiments. RESULTS

Studies in Mouse Blastocysts No binding for All above the nonspecific range was found in early mouse embryos under a number of experimental conditions, including variations in temperature, time, number of embryos, and method of separation of bound and free radioactivity. Attempts to measure All binding in membrane fractions of blastocyst were also unsuccessful. In contrast, AII binding was observed in membranes prepared from later gestational embryos and subsequent efforts were made to characterize the nature of this binding.

Properties of All Binding in Late Gestation Conceptus Initial binding studies in membranes from whole mouse and rat fetuses at 17 to 19 days of gestation showed very high levels of specific high affinity All binding. To determine how much of this binding activity in the whole fetus was due to adrenal, liver and visceral smooth muscle All receptors, binding experiments were performed in rat fetuses in which all visceral organs including the brain were removed. As shown in Fig. 1 the amount of binding on a mg of protein basis was slightly higher in membranes from eviscerated fetuses, indicating that both visceral and nonvisceral tissue have significant binding sites for All. Further characterization of All binding was performed on membrane-rich fractions prepared from eviscerated rat fetuses. Binding was linear with increasing protein concentrations up to 100 I~g. Using between 30 to 70 Ixg of protein, the binding was time and temperature dependent, reaching equilibrium at 45 min and maintaining a plateau for an additional 90 min at 23°C. At 37°C equilibrium binding was reached in 30 min, but was less stable showing a decrease by 120 min (Fig. 2). Binding was enhanced in the presence of the divalent cations Mg z+ and Ca 2+, reaching a maximum 20% increase with 2.5 mM Mg 2÷ and 15% increase with 10 mM Ca2+ . Increasing sodium concentration from 10 to 100 mM resulted in slight inhibition of the binding. Dithiothreitol caused a concentration-dependent increase in specific binding to a maximum of 25% with 2.5 mM addition. A similar 20% enhancement was observed with 2 mM EGTA, while bacitracin and trypsin inhibitor had little effect and phenylmethylsulfonyl fluoride (0.1 mM) was inhibitory. The specificity of the binding for AII was indicated by the

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ability of AII analogues to inhibit the binding of ~zsI[Sar~,IleS]AII to fetal membranes (Fig. 3). In 3 experiments in 18-day-old fetuses, the native peptide All and AIII had similar potencies with IC5o values of 2.4 ± 0.4 and 3.2--_0.6, respectively. The superagonist [Sar~]AlI and the antagonist [Sar ~,IleS]AII were more potent with IC5o values of 0.84 ± 0.1 and 0.8 ± 0.1 nM, respectively. AI was less potent with an IC5o of 14.3±2.9 nM, while the All nonrelatedpeptides, CRF, GnRH, VP, ACTH(I-24) and ACTH(139) had no inhibitory binding activity (Fig. 3). Similar binding specificity was observed in membranes from 15-day-old fetuses. Scatchard analysis of the binding data at equilibrium showed a single type of high affinity sites with Kd ranging betwen 0.7 and 0.9 nM, while binding capacity was dependent on gestational age (Fig. 4). Binding was first detectable about day 10 (14.3+_2.3 fmol/mg), increasing to 104 ± 16, 2,625 ± 168, 5,993 +_152 and 5,902±92 by days 12, 15, 18 and 21 of gestational age, respectively. The concentration of sites at day 15 was similar to that found in adrenal glomerulosa membranes of adult rats but doubled by day 18. As shown in the figure, no changes in binding affinity were observed with increasing gestational age. For these binding sites to have physiological significance it would require the presence of the appropriate ligand, All. Estimation of the presence of All in eviscerated fetal extracts by radioimmunoassay indicated relatively high amounts of immunoreactive All-like material of 845 pg/g of tissue, levels higher than those described for plasma in adult sodium restricted rats (200 pg/ml) (1). The identity of the immunoactive All was confirmed by chromatogrpahic elution pattern and its ability to displace radiolabelled All analogs from adrenal membranes. The material from fetal extracts eluted as a single peak of All immunoactivity which coincided with the position of an All standard subsequently aplied to the same column. A peak of radioactivity corresponding to the 125I-AII initially added to correct for losses was found shifted to the right, but overlapping the peak of AII (Fig. 5). Since the resolution of the Sephadex column does not allow separation of All from AIII, which is equally recognized by the antibody, the immunoactive material may include both peptides. The AII bioactivity of the immunoreactive All is shown in Fig. 6. Addition of 50 and 100 i,tl aliquots of the material pretreated with normal rabbit serum displaced the binding of ~=5I[Sarl,IleS]AII to adrenal glomerulosa membranes with activity consistent with the All concentration measured by RIA (5.2 ng/ml). Pretreatment of the

462

JONES, MILLAN, NAFTOLIN AND AGUILERA

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FIG. 6. Biological activity of immunoactive AII extracted from rat fetuses as shown by its ability to displace ~25[Sart,IleS]AII from rat adrenal glomerulosa membranes. Arrows indicate the addition of aliquots of extract pretreated with normal rabbit serum (~) and with All antibody (A).

extract with All antibody abolished its binding activity to the adrenal membranes. DISCUSSION

This report describes the presence of high concentrations of AII binding sites in rat fetuses during the second half of intrauterine development. Since All has been shown to accelerate the rate of blastocyst development (22), attempts were made to detect All binding during this very early stage. Although not successful, it is possible that the receptor concentration was below the sensitivity of the assay. Since the blastocysts consisted of only 70 cells and the tissue yield was critically low it is not possible to rule out that the receptors are present at these stages. Similar to the present observation in the rat, abundant All binding sites have been shown in late gestation mouse fetuses (35). In the studies in the rat, the first age when receptors could be detected reproducibly (n = 3) was day 10. However, at day 10 and 12 the embryo could not be separated from the embryonic membranes and it is possible that some binding is present in these membranes or that a higher level of binding in the embryo was diluted by the presence of the membranes. By day 15 when organogenesis is complete, All binding capacity in the eviscerated fetus was comparable to that observed in rat adrenal glomerulosa, tissue known to contain the highest concentration of functional All receptors in the adult (10,15). The concentration of AII binding sites continued to increase, reaching more than twice the levels in the adrenal at 18 and 20 days of gestational age. The studies in eviscerated fetuses were initially performed on the assumption that most of the binding sites were confined to visceral organs such as adrenal, kidney, liver and smooth muscle of the gut. It was significant therefore that very high levels of binding were observed in the eviscerated fetus, strongly implicating the presence of All recpetors in such tissues as skeletal muscle, cartilage, skin, and connective mesenchymal tissue. The location of AII receptors in mesenchymal tissue and skeletal muscle has recently been confirmed by autoradiographic studies in our labo-

ratory (35). The characteristics of the fetal All binding sites are similar to those described for All receptors in recognized target tissues in the adult. It is interesting to note that in contrast with other tissues, in which AI is 100 times less potent than All, in the fetus it is only 6-times less active. Since these values were obtained in the presence of EGTA, which inhibits converting enzyme, the higher potency is due to intrinsic higher activity of the decapeptide and not to conversion to All. In developing a case for the possible function of these All binding sites there are several lines of evidence to be considered. Firstly, the extensive characterization of the binding sites, as reported in here including specificity, affinity, and ionic dependence, indicate that the binding properties are similar to those of All receptors in the adrenal glomerulosa and other target tissues for All action. In addition, it was possible to detect substantial amounts of bioactive angiotensin, thus providing a ligand to act through these binding sites to mediate some physiological effect. All components of the renin-AII system including renin (6, 28, 30, 34, 35), angiotensinogen (20) and converting enzyme (32,33), are present in the fetal placental unit. Therefore, the peptide is likely to be formed at one or more sites in the fetus, such as the placental compartment, fetal circulation from fetal kidney renin, or local tissue sites. The visceral binding is likely associated to recognized target tissues such as the adrenal gland and kidney, where as in the adult, AII may have a role in the regulation of steroidogenesis and hydroelectrolyte metabolism (10). The most intriguing question concerns the possible function of the All binding sites in nonvisceral tissue. It is logical to assume that this binding represents receptor sites for the reasons indicated above, and the widespread distribution of these receptors is compatible with some general effect of All on the growth and differentiation of the fetus. It was not possible to recognize specific binding on early mouse embryo and up to day 10 of gestation in the rat. However, All receptors were found from day 10 onward. On day 10 the rat embryo is at the neurula stage, a disc like structure astride the yolk sac with 0 to 4 somites. During the next 4 days (day 11 to 14), the embryo will pass through most of organogenesis, consisting of about 50 somites and appearing as a fetus with a complete integument, limb buds and floating within the ammiotic sac, tethered by the umbilical cord and vitalline vessels (16). All receptor sites develop rapidly during organogenesis and the most striking increase in All binding was observed between days 15 and 18 when fetal growth, including rapid development of skeletal muscle and skin, is the predominant event. The possibility that All may have a role in cellular growth has been suggested by recent experiments showing that AII increased DNA synthesis, PDGF receptor and expression of the growthrelated oncogenes, c-fos and c-myc, in cultured smooth muscle cells (3, 19, 25). It has also been postulated that AI! may have a role in angiogenesis based on experiments in the rabbit showing an increase in corneal vascularization by All (13). Although the specificity of such effects remains to be proven, it is possible that All may act as an angiogenic factor during fetal development. Further studies are clearly needed to determine the exact cellular localization and coupling of these fetal All binding sites to a cellular function. However, the characteristics of the binding in conjunction with the presence of considerable amounts of bioactive angiotensin in the fetus suggest that these sites are functional receptors for AII and support a unique role for the octapeptide during fetal development.

AII R E C E P T O R S IN R A T FETUS

463

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20.

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35.

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