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c Instituto de Histología y Patología, Universidad Austral, Valdivia, Chile
d Centro de Investigaciones Médicas, P. Universidad Católica, Santiago, Chile
e Institut für Biochemie II, Universitäts Krakenhaus Frankfurt, Frankfurt 6230, Germany
f Departamento de Nefrología, P. Universidad Católica, Santiago, Chile
ABSTRACT
Tissue kallikreins are present in rat uterus during the estrous cycle in luminal and glandular epithelium, in early gestation in the implantation node, and in the last third of pregnancy surrounding the sinusoids in the decidua basalis. The pattern of kinin B2 receptor expression, through which the vasoactive effect of kallikreins is exerted, was studied by in vitro autoradiography and immunohistochemistry. The kinin B2 receptor was observed in the luminal and glandular epithelium, myometrium, endothelial cells of arteries, veins and venules, and smooth muscle cells of endometrial and myometrial arterioles. Immunoblotting of crude membranes revealed a band of 69 kDa that increased in late proestrus and estrus, concordantly with the pattern of immunostaining observed in the tissue. At Day 7 of gestation, the kinin B2 receptor was expressed (binding sites and receptor protein) in the epithelium of the implantation node and decidual cells; these latter cells showed a further increase during gestational Days 9 and 10. From Days 14 to 21, the subplacental decidua became strongly immunoreactive, and on Days 16 and 21 the placental labyrinthine endothelium was intensely stained. During this period, endothelium of arteries and veins, smooth muscular cells of small diameter arterioles, and myometrium also expressed B2 receptors. In unilaterally oil-stimulated pseudopregnancy, the decidual cells and the glandular epithelium show similar immunoreactivity to that during pregnancy. The temporospatial pattern of kinin B2 receptors, coinciding with that of kallikrein or with sites accessible to the generated kinins, further supports an autocrine-paracrine role for the kallikrein-kinin system in the vasoactive changes of implantation and placental blood flow regulation.
implantation/early development, placenta, pregnancy
INTRODUCTION
Kinins, the vasoactive nona- and decapeptides generated from kininogens by the enzymatic action of kallikreins exert their biological actions by stimulating at least two types of receptors, B1 and B2 [1]. Expression of the B1 receptor becomes evident during the inflammatory response [2, 3], whereas the B2 receptor is constitutively expressed on many tissues and organs and mediates most of the effects induced by kinins, such as the increase in local blood flow and vascular permeability, hypotension, and pain. In the estrous cycle bradykinin increases uterine blood flow [4], while in pregnancy bradykinin and angiotensin-converting enzyme inhibitors that decrease bradykinin degradation and enhance tissue content increase uteroplacental blood flow, an effect that is blocked by bradykinin antibodies [5]. Trasylol, a serine protease inhibitor, administered to rats in late pregnancy prolongs parturition [6]. The responsiveness of the rat uterus to bradykinin in vitro has been used as a bioassay of kallikrein activity [7]. These findings support a role for the kallikrein-kinin system, and especially for kinin B2 receptors, in the modulation of fetoplacental perfusion and myometrial contractility.
The various components of the kallikrein-kinin system such as kallikreins [8–10], kininogens [9], and kinin B2 receptors [11–14] are expressed in the rat uterus. Kallikreins have been demonstrated in the luminal and glandular epithelial cells of rat uterus during the estrous cycle. During early gestation, immunoreactivity increases in the implantation node, whereas between Days 16 and 21 of pregnancy kallikrein staining surrounds the sinusoids of the whole decidua basalis, a distribution pattern clearly related to areas involved in implantation and placental blood flow regulation. Binding and/or B2 receptor immunoreactive protein is present in both myometrium and endometrium of the various species investigated. We have previously immunolocalized the B2 receptor in the luminal epithelium, in endometrial glands, and in myometrial cells of the nonpregnant rat uterus [13]. Distribution of the kinin B2 receptor has also been demonstrated in the uterus of the rat, guinea pig, and sheep [14–16]. Furthermore, myometrial B2 receptor binding and mRNA levels are increased during late proestrous and in ovariectomized estrogen-supplemented rats [14].
To advance further in the understanding of the uterine kallikrein-kinin system, especially under the marked vasoactive challenge of gestation, the present study examines the distribution of the kinin B2 receptor during the estrous cycle, pregnancy, and pseudopregnancy by using in vitro receptor autoradiography and immunohistochemistry.
MATERIALS AND METHODS
Materials
The following materials were obtained from commercial sources: Swine antirabbit IgG, peroxidase/antiperoxidase complex (Dako, Carpinteria, CA); Tris, Triton X-100, Tween-20, 4-chloronaphthol, BSA, polylysine, histology-grade gelatin, nitrocellulose membranes, Tyr0-bradykinin, bradykinin, SDS, polyacrylamide, ß-mercaptoethanol, PMSF (Sigma Chemical Co., St. Louis, MO); sodium meta-periodate, L-lysine monohydrochloride, NaHCO3, and Histosec (Merck, Darmstadt, Germany); aprotinin, leupeptin, pepstatin, and antipain (Roche Diagnostics GmbH, Mannheim, Germany); immunoassay-grade gelatin (Bio-Rad, Richmond, CA); autoradiographic emulsion L4 (Ilford, England, UK); autoradiographic film Biomax MR-1 (Kodak, Rochester, NY); [125I]NaI (specific activity 16 Ci/mg, Comisión de Energía Nuclear, Chile). Rabbit antiserum against purified rat urinary kallikrein was kindly donated by Prof. Hector Croxatto (Catholic University, Santiago, Chile). The kinin B2 receptor antagonist HOE140 (Icatibant) was kindly donated by Dr. B. Schölkens (Hoechst Marion Roussel, Frankfurt am Main, Germany).
Animals
Adult female Sprague-Dawley rats weighing 200–250 g were housed in a light- and temperature-controlled room, with free access to chow and water. Vaginal smears were taken daily. Rats presenting a regular 4-day cycle were randomly assigned to two experimental groups: estrous cycle and pregnancy. Rats allotted to estrous cycle continued being subjected to daily vaginal smears. Rats assigned to the pregnancy group were caged with fertile males on the night of proestrus. The presence of sperm in the vaginal smear defined Day 1 of pregnancy. In a third group of animals, pseudopregnancy was attained by mating on the night of proestrus with vasectomized males. Day 1 of pseudopregnancy was certified by the presence of a vaginal plug and by the sustained leukocytosis of metestrus in subsequent vaginal smears. Luminal stimulation was performed on Day 5 under light ether anesthesia, by the instillation of 0.05 ml sesame oil into one of the horns between 1000 and 1030 h, using a 5-cm 24-gauge blunt needle connected to a 250-µl Hamilton syringe, after visualizing the cervix through an otoscope. All research animals were handled in compliance with the NIH Guide for Care and Use of Laboratory Animals.
Tissue Collection
Four to 10 animals were killed between 1000 and 1200 h at diestrus, early proestrus, late proestrus, estrus, Days 5, 6, 7, 9, 10, 12, 14, 16, and 21 of pregnancy (P5–P21) and on Day 7 of pseudopregnancy, 48 h after unilateral intraluminal stimulation. Rats were anesthetized with nembutal, perfused with saline through the abdominal aorta for 2 min, and then with 2 ml of periodate-lysine-paraformaldehyde fixative (2% paraformaldehyde, 2.2% sodium metaperiodate prepared in 0.1 M lysine-monohydrochloride-phosphate buffer, pH 7.4) [13, 17]. After perfusion, the uterine horns were immersed in fixative at room temperature for 48 h and then dehydrated in a graded series of ethanol and embedded in Histosec. Uteri were sectioned both sagittally and longitudinally throughout the different stages of gestation. Four to five sections (5 µm thick) of different sites were analyzed for each animal; these were mounted on glass slides coated with polylysine.
Immunoblotting
Crude membranes were prepared from animals killed under ether anesthesia. Uterine horns from rats at diestrus, early proestrus, late proestrus, and estrus were removed and immediately washed in freshly prepared Hanks buffered saline solution (HBSS). The tissues were placed in cold HBSS, stripped of fat and surrounding connective tissues, cut in small pieces, and homogenized in 1 mM NaHCO3 containing 0.1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 0.1 µg/ml antipain, and centrifuged at 13 000 x g for 45 min at 4°C. The pellets were resuspended (100 µg of protein per lane) directly in electrophoresis sample buffer, run on 12.5% SDS-PAGE in the presence of 5% ß-mercaptoethanol, and electrotransferred to nitrocellulose [18]. Blots were incubated overnight with antiserum AS351 (1:100) to a synthetic peptide MLN33 of the extracellular domain-1 (ED1) of the kinin B2 receptor [19]. Characterization and specificity of this antibody have already been established [13, 19]. Bound antibody was visualized by an 125I-labeled secondary antibody or by the peroxidase/antiperoxidase method [20]. Nonspecific immunoreactivity was determined by replacing the antipeptide antiserum by preimmune serum at the same dilution. The intensity of the bands was quantified by the Un-scan-it gel automated digitizing system (asknet; Karlsruhe, Germany). Results were expressed as mean ± SEM. Significance was established by the Student t-test and considered acceptable at the 5% level (P < 0.05).
In Vitro B2 Receptor Autoradiography
The bradykinin analogue Tyr0-bradykinin was radioactively labeled with [125I]NaI (specific activity 16 Ci/mg; Comisión de Energía Nuclear, Chile) [21]. Freshly removed uterine horns were rapidly frozen in liquid nitrogen, sections of 15–20 µm were prepared in a cryostat at -30°C, mounted on slides precoated with gelatin, and stored at -80°C. Before use, the slides were warmed to room temperature and washed with cold 50 mM Tris-HCl, pH 7.4 [22], containing 0.1% BSA, 1 mM EDTA, 1 mM EGTA, 20 µM captopril (washing buffer). Incubation was done at 4°C overnight with 5 nM [125I]Tyr0-bradykinin dissolved in washing buffer containing 1 mM dithiothreitol, 140 µg/ml bacitracin, and 10 µM DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The sections were rinsed with cold washing buffer, distilled water, rapidly cool-dried, and exposed to a Biomax MR-1 film for 72 h at -20°C. Binding specificity was probed in the presence of 5 µM of unlabeled Tyr0-bradykinin, bradykinin, or HOE140 (Icatibant).
Immunostaining Procedure
The immunostaining technique used was the unlabeled antibody method of Sternberger modified as previously described [13]. Sections prepared from tissue fixed in periodate-lysine-paraformaldehyde were dewaxed with xylene, rehydrated through a graded series of ethanol, and treated with absolute methanol and 1% hydrogen peroxide to block endogenous pseudoperoxidase activity [21]. After rinsing (3 x 5 min) in 50 mM Tris-HCl buffer, pH 7.8, for 5 min, the sections were incubated sequentially with 1) a mixture of eight polyclonal rabbit antisera (1:500–1:1000) raised against peptide sequences of the extracellular and intracellular domains of the rat kinin B2 receptor for 18 h. This mixture was used in order to achieve a maximal immunoreactivity for the B2 receptor protein in the tissue. Characterization and specificity of each peptide antibody have already been established [19, 21]; 2) swine antirabbit IgG (1:80) for 30 min; and 3) peroxidase/antiperoxidase complex (1:100) of rabbit origin for 30 min. All incubations were performed at 22°C in a water bath used as a humidifier chamber. Between incubations the sections were washed (3 x 5 min) in Tris-HCl buffer, pH 7.8. The antisera and peroxidase/antiperoxidase complex were diluted in 50 mM Tris-HCl buffer, pH 7.8, containing 1% immunoglobulin-free BSA. Peroxidase activity was demonstrated with 0.1% 3-3'-diaminobenzidine and 0.03% hydrogen peroxide for 15 min at room temperature. To compare the topographical relationship of kinin B2 receptors with the kinin-generating enzyme, we also used a polyclonal antiserum raised in rabbits against purified rat urinary kallikrein (1:1000). The cross-reactivity of this antiserum assessed by dot-blot immunoassay was strong for rK1, moderate for rK2, and rK7 and minimal for rK9; the kininogenase activity is mainly displayed by rK1, being weak for rK7 [10].
Immunohistochemical controls were prepared by omission and/or replacement of antipeptide antibodies by preimmune rabbit sera and by incubating tissue sections with the antipeptide antibodies in the presence of an excess (50–100 µg/ml) of the same peptides used for immunization [13].
RESULTS
Binding of Iodinated Bradykinin Versus Immunoreactivity to Antipeptide Antibodies
The detection of kinin B2 binding sites by in vitro autoradiography was largely congruent with the pattern of immunostaining obtained by antipeptide antibodies (Fig. 1). Both methods demonstrated the presence of binding sites/immunoreactive receptor protein in myometrium and endometrium. In myometrium the kinin B2 receptor was expressed in the inner and outer muscular layers (Fig. 1, a, b, d, and e), whereas in endometrium its distribution was mainly associated with the luminal epithelium and endometrial glands (Fig. 1d). In addition, immunoreactivity was intense in the smooth muscle cells of arterioles present at both sites (Fig. 1e). An excess of unlabeled bradykinin or HOE140 displaced the binding of the radioactive peptide (Fig. 1c), and immunoreactivity was abolished when an excess of the same peptides used for immunization was mixed with the antisera during incubation of the sections (Fig. 1f).
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Estrous Cycle
Immunoblotting of crude membranes prepared from various stages of the estrous cycle, immunoprinted with an antipeptide antibody directed to the amino terminus of the kinin B2 receptor, revealed a unique immunoreactive band of 69 kDa. The relative amount of immunoreactive B2 receptors present was higher in late proestrus and estrus than in diestrus and early proestrus (Fig. 2). Replacement of the antipeptide antibody by preimmune serum did not reveal any immunoreactivity (Fig. 2).
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Tissue immunostaining during the estrous cycle was present at the luminal and glandular epithelial cells, in the inner and outer muscular layers, and in the wall of arterioles (smooth muscle cells; Figs. 1e and 3b) and venules (endothelial cells; Fig. 3b). The intensity of the immunoreactivity was mild in diestrus (Fig. 3a) and early proestrus (not shown), strong in late proestrus (Fig. 3b), and moderate in estrus (Fig. 3c). Differences in the intensity of the staining were mainly associated with the immunoreactivity expressed by luminal epithelium and glands (Fig. 3).
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Replacement of the antipeptide serum mixture by nonimmune serum or immunocompetition with an excess of the same peptides used for immunization resulted in the absence of relevant immunostaining (Fig. 3, d and e).
Colocalization of Kinin B2 Receptors and Kallikreins in Endometrial Gland Cells
Independently of the intensity of immunostaining, during the estrous cycle (Fig. 3) and at the beginning of pregnancy, kinin B2 receptors coexisted in glandular cells with uterine kallikreins, which in our previous study [10] were identified as rK1 and rK7 (Fig. 4). Nevertheless, these two components displayed a distinctive subcellular distribution; while immunoreactivity for kinin B2 receptors was intense on the basolateral and luminal epithelial cell membrane (Fig. 4, a and b), the kallikreins were usually expressed in the cytoplasm of epithelial glandular cells (Fig. 4c).
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Pregnancy
Between Days 5 and 7 of gestation, luminal and glandular epithelial cells were labeled, achieving greater intensity in the luminal foldings; the smooth muscle cells of uterine arterioles were also stained. At Day 7, an intense binding of the iodinated bradykinin was demonstrated by autoradiography, and positive immunostaining was observed in the epithelial cells of the implantation node, in the glandular epithelium, and in decidual cells of some specimens (Fig. 5, a and c). Immunohistochemistry demonstrated that reactivity to antipeptide antibodies in decidual cells was clearly related to the cell membrane, though some granular staining was also present in the cytoplasm (Fig. 5c). The intensity of the staining in the implantation chamber and in the glandular epithelium is comparable in intensity to that observed in late proestrus.
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At Days 9 and 10 of pregnancy, the initial stage of placentation, the number of decidual cells expressing kinin B2 binding sites (Fig. 6a), and the receptor protein (Fig. 6, c and d) increased markedly in comparison with Day 7 of pregnancy and with that observed in a few atrophied glands.
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From Days 14 to 21 the subplacental decidual cells became strongly immunoreactive (Fig. 7b). On Days 16 and 21, the endothelial cells present in the labyrinthine placenta were intensely stained for kinin B2 receptors (Fig. 7, b and d), whereas the decidua basalis (Fig. 7a) and the basal placenta were devoid of any significant staining. In addition, immunoreactive B2 receptors were usually expressed by endothelial cells of muscular arteries and veins (Fig. 7e) and by smooth muscular cells of small diameter arterioles (Fig. 7f).
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Immunostaining was also present in the inner and outer muscular layers, showing a decreased reactivity in comparison to the estrous cycle (not shown).
Independently of the period of gestation analyzed, immunostaining and binding were displaced by an excess of the peptides used for immunization (Figs. 5d, 6e, and 7c) and by an excess of the kinin B2 antagonist HOE140 (Figs. 5b and 6b).
Stimulated and Nonstimulated Pseudopregnancy
The horn submitted to oil-induced decidualization showed, in Day 7 of pseudopregnancy, immunoreactive B2 receptors associated to the surface and cytoplasm of decidual cells. The staining of the luminal epithelium was similar in the stimulated and the nonstimulated horn, while the uterine glands of the decidualized horn had an increased immunoreactivity, as compared to the contralateral one (Fig. 8, a–e).
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DISCUSSION
The present study provides new information regarding the distribution pattern of kinin B2 receptors in the rat uterus during the estrous cycle, throughout pregnancy and in unilaterally intraluminally stimulated pseudopregnacy. During the estrous cycle, autoradiography (binding of iodinated bradykinin) and immunohistochemistry (immunoreactive receptor protein) demonstrated the existence of kinin B2 receptors in the luminal epithelium, the epithelial cells of uterine glands, in stromal cells, and in both muscular uterine layers. Additionally, the sensitivity provided by immunohistochemistry allowed us to identify the kinin B2 receptor protein in vessels of small diameter, like arterioles and venules. Immunoblotting performed on crude membrane extracts indicated that the rat uterine B2 receptor has a molecular mass similar to that reported in human fibroblasts [19, 23], myometrium [24], and kidney cells [13, 25]. By using this dual approach we observed that kinin B2 receptors were more abundant during late proestrus and estrus than diestrus and early proestrus. Modulation of kinin B2 receptor levels during the estrous cycle is probably related to estrogen levels, because estrogens peak during late proestrus. Previous reports have shown that kinin B2 receptor levels in both endometrium and myometrium are lowest during early proestrus, when estrogen levels are low, whereas myometrial B2 receptor protein and mRNA together with estrogen levels are highest during late proestrus [14].
Although it has been shown that decidual cells express functional kinin B2 receptors and that their stimulation induces arachidonic acid release [26, 27], this is the first study to demonstrate precisely the nature of B2 receptors in these cells, because the binding and immunoreactivity was confirmed by displacement of the labeled peptide by HOE140/unlabeled bradykinin and by competition of the antipeptide antibodies with the same peptides used for immunization. Decidual cells in pregnancy and oil-induced deciduomata clearly bound radiolabeled bradykinin and displayed most of the immunoreactivity on their cell membrane. The similar expression of the B2 receptors in decidual cells and in glandular epithelium in pregnancy and oil-stimulated pseudopregnancy shows that both the blastocyst and oil droplets are able to induce the B2 receptor in these cell types, suggesting that the topical stimulation of the luminal epithelial surface (shear stress or contact) represents a more potent trigger to the underlying tissues than the hormones in the maternal milieu or those derived from the embryo.
In several of the periods studied, the glandular and luminal epithelium and the cells that surround the subplacental sinusoids express both kinin B2 receptors and kallikreins [10]. This particular spatial distribution of kallikreins and kinin B2 receptors provides a structural basis for an autocrine action of kinins on the same cells that contain the kinin-releasing enzyme, or for a paracrine effect on the neighboring cells. Coexistence of the enzyme and/or kininogens (kinin-containing substrate) with kinin B2 receptors have also been found in other rat organs such as the kidney [21] and the salivary gland [23]. On the other hand, isolated expression of kinin B2 receptor protein and binding sites is present in the decidua at Days 7, 9, and 10, in the subplacental decidua, and in the placenta in the second half of pregnancy. During decidualization, kinins generated by the action of glandular kallikreins and trapped circulating kininogens could attain the secondary decidual zone through gap junctions demonstrated in these areas. At Days 9 and 10, when uterine kallikreins are almost absent [10], kinins could be generated from circulating kallikreins and kininogens. In established placentation, kallikreins synthesized in the subplacental sinusoids could act on circulating kininogens, and kinins could seep to the placental bed.
The physiological significance of kinin B2 receptors in endometrium has not been clarified yet, though it has been shown that bradykinin induces in vitro proliferation of endometrial stromal cells [28], enhances sodium absorption in glandular epithelial cells [29], and stimulates prostaglandin synthesis in both glandular and stromal cells [30]. Interestingly, during early pregnancy prostaglandin synthase is also located in the luminal and glandular epithelium, in stromal cells adjacent to the luminal epithelium, in the secondary decidual zone, and in blood vessels [31]. Furthermore, cyclooxygenase (COX)-1 and COX-2 are localized primarily in epithelial cells of the endometrium and in the myometrium, both increase during pregnancy and parturition, and COX-2 is elevated in proestrus and estrus [32]. On the other hand, uterine constitutive endothelial nitric oxide synthase (NOS III) is abundantly expressed in luminal and glandular epithelium, in myometrium, and in endothelial cells, during proestrus and estrus, periods in which immunoblot analysis demonstrates high levels of kinin B2 receptors [33, 34]. NOS III uterine levels remain stable throughout pregnancy, while NOS II (inducible NOS) increases in gestation and decreases during delivery [35]. The coincidence between the temporospatial pattern of kinin B2 receptors and of enzymes responsible for the synthesis of prostacyclin and nitric oxide point to a close interrelationship between these uterine vasodilator mechanisms. In fact, in cultured human decidual cells and in cultured trophoblast, bradykinin induce prostacyclin and NOS, respectively [26, 36].
In conclusion, our results demonstrate kinin B2 receptors in cell types and tissues previously shown to respond to bradykinin stimulation and to synthesize the interrelated vasodilators prostacyclin and nitric oxide. This evidence, together with previous reports on the existence of various components of the kallikrein-kinin system in the uterus in sites that establish the first embryo-maternal contact, and later undergo important vasoactive changes, speak in favor of a meaningful role for this system in the mechanisms of embryo and trophoblast attachment as recently suggested [37], and in the regulation of the vasoactive and trophic changes that occur during gestation.
ACKNOWLEDGMENTS
We appreciate the expert technical assistance of Mr. C. Lizama, Mr. U. Novoa, Mr. J. Sarmiento, and are very grateful to Dr. B. Schölkens (Hoechst-Marion Roussel, Frankfurt Am Main, Germany) for donating HOE140.
FOOTNOTES
First decision: 1 September 2000.
1 This work was supported by grant 1980958 from FONDECYT (Chile), by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and the Volkswagen Foundation (Germany). 
2 Correspondence: Gloria Valdés, Departmento de Nefrología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Diagonal Paraguay 361, Torre 10, 1er Piso, Santiago, Chile. FAX: 562 639 7377; gvaldes{at}med.puc.cl
Accepted: January 22, 2001.
Received: July 24, 2000.
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