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Endocrinology Vol. 140, No. 7 3372-3382
Copyright © 1999 by The Endocrine Society

ARTICLES

Localization of Bradykinin B2 Receptors in the Endometrium and Myometrium of Rat Uterus and the Effects of Estrogen and Progesterone1

C. Murone, S. Y. Chai, W. Müller-Esterl, F. A. O. Mendelsohn and J. Clements

Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne (C.M., S.Y.C., F.A.O.M.), Parkville, Victoria 3052, Australia; Institute of Physiological Chemistry and Pathobiochemistry, Johannes-Gutenberg University (W.M.-E.), Mainz D-55099, Germany; and the Center for Molecular Biotechnology, School of Life Sciences, Queensland University of Technology (J.C.), Brisbane, Queensland 4001, Australia

Address all correspondence and requests for reprints to: Dr. C. Murone, Department of Anatomical Pathology, Austin and Repatriation Medical Center, Level 6, Harold Stokes Building, Austin Campus, Heidelberg, Victoria 3084, Australia. E-mail: carmel.murone{at}ludwig.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the uterus, bradykinin is a potent inducer of smooth muscle contraction, which is mediated by the bradykinin B2 receptor subtype. However, little is known about the distribution or regulation of this receptor in this tissue. The aim of this study was to localize the B2 receptor in the uterus and determine whether the levels of this receptor were altered during the estrous cycle and modulated by estrogen and/or progesterone in ovariectomized rats.

At diestrus, uterine B2 receptors were localized to both the circular and longitudinal smooth muscle layers of the myometrium, the endometrial stroma, the glandular epithelium, and the layer subjacent to the luminal epithelium. B2 receptor levels in both myometrium and endometrium were lowest during early proestrus, when estrogen levels are low, whereas myometrial B2 receptor protein and messenger RNA levels were highest during late proestrous, when estrogen levels peak. Similar findings were observed for the estrogen-supplemented group after ovariectomy, with progesterone appearing to inhibit the estrogen-induced rise in bradykinin B2 receptor density in estrogen/progesterone-treated animals.

Using in vitro receptor autoradiography employing the specific B2 receptor antagonist analog, HPP-HOE140, immunostaining with specific antipeptide antibodies generated against the B2 receptor, and in situ hybridization using a specific bradykinin B2 receptor riboprobe, our findings show a discrete distribution of the bradykinin B2 receptor throughout the different layers of the uterus and suggest that bradykinin B2 receptor levels in the rat uterus are regulated by estrogen, and possibly progesterone, in both myometrium and endometrium.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BRADYKININ is a nonapeptide formed by the action of kallikrein on kininogen and has been implicated in the mediation of cardiovascular homeostasis, pain production, and inflammation (1). The involvement of kinins in vasodilation and expansion of fluid volume is closely associated with the pathogenesis of inflammation (1), processes that have also been implicated in the reproductive events of embryo implantation and ovulation (2, 3, 4). Bradykinin is also involved in follicular contraction at ovulation (5), uterine smooth muscle contraction at parturition (6, 7), as well as maintaining uteroplacental blood flow in established gestation (8, 9). In addition, a kinin that exists only in the rat, T-kinin, and that is thought to be involved primarily in the inflammatory response, has properties similar to those of bradykinin, in that both peptides contract rat uterus by acting on the bradykinin B2 receptor (10).

Most of the components of the kallikrein-kinin system are expressed in the female reproductive tract, ovary, uterus, and placental tissues (6, 8, 11, 12, 13, 14). In the rat uterus, tissue kallikrein activity, kininogen levels, and immunolocalization of tissue kallikrein have been shown to change during the estrous cycle and pregnancy (13, 14, 15). Given the roles attributed to kinins in the reproductive tract, it is surprising that little is known about the cellular distribution and regulation of its receptors, particularly the B2 receptor subtype, which has been suggested to modulate the myostimulating activity of rat tissue kallikrein in the rat uterus (16).

We have previously used in vitro receptor autoradiography to localize the B2 receptor in the endometrium and myometrium of the guinea pig and sheep uterus (17, 18). Bradykinin or the B2 receptor has also been immunolocalized to the endometrium and myometrium of the rat and human uterus (19, 20). Apart from these studies, there is little information on the spatio-temporal distribution of the B2 receptor subtype in the uterus across the estrous or menstrual cycle or its regulation by estrogen and progesterone.

The aim of this study was first to confirm the cell-specific localization of B2 receptors in the rat uterus by these different, but complementary, methods: in vitro receptor autoradiography using 4-hydroxyphenyl-propyl-D-Arg[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (HPP-HOE140), an analog of the specific B2 receptor antagonist, D-Arg[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (HOE 140), in conjunction with in situ hybridization histochemistry and immunohistochemistry. Second, we wished to determine whether uterine B2 receptor levels change throughout the estrous cycle and are modulated by estrogen and/or progesterone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Timing of the estrous cycle
Female Sprague Dawley rats (160–190 g; n = 20) were housed in a light- and temperature-controlled room, with free access to chow and water. Vaginal smears were taken daily to assess the stage of the estrous cycle, and four to six animals were killed between 1200–1400 h at either diestrus, early proestrus, late proestrus, or estrus. Tissues from two animals in the estrus group were lost giving a final n = 2 for that group.

Ovariectomy and steroid supplementation
Adult female rats (160–190 g; n = 4–6/group) were anesthetized by an im injection of ketamine (75–95 mg/kg; Ketamar, Mavlas, Sydney, Australia) and xylazine (5–8 mg/kg; Rompun, Bayer, Sydney, Australia) mixture, and both ovaries were removed via an incision on the dorsal side at the level of the second lumber spinal segment. The incision was sutured, and the animals were allowed to recover. Forty-eight hours postsurgery, estrogen (2 µg; estradiol benzoate, Intervet, Castle Hill, Australia) and/or progesterone (2 mg; Intervet) dissolved in 0.1 ml maize oil was administered sc daily for 5 days. Control groups comprised of untreated sham-operated animals and ovariectomized animals injected with oil alone. Two animals died in the progesterone-treated group, giving a final n = 3 for these two groups.

Blood and tissue collection
Rats were killed by carbon dioxide inhalation. Trunk blood was collected to determine serum estradiol and progesterone levels using commercially available specific RIA kits (estradiol: DiaSorin, Inc., Sorin Diagnostics, Saluggia, Italy; progesterone: AmerlexP, Amersham International, Aylesbury, UK). Uterine horns were removed, frozen in isopentane on dry ice, and stored at -80 C.

All animal experiments were approved by Queensland University of Technology ethics committee, which adheres to the Australian Code of Practice as set out by the National Health and Medical Research Council of Australia.

Localization of bradykinin B2 receptors and competition binding analysis by in vitro receptor autoradiography
In vitro autoradiography and competition binding analysis were performed as described previously (17, 21, 22). Briefly, tissue sections (10 µm) were cut on a cryostat at -20 C and thaw-mounted onto gelatin-chrom alum-coated slides. Sections were preincubated in 170 mM Tris-HCl buffer (pH 7.4) containing 0.2% BSA at 22 C for 15 min. This was followed by a 24-h incubation at 4 C in a fresh volume of the same buffer containing 0.28 µCi/ml of the B2 receptor antagonist analog, [125I]HPP-HOE140 (~200 pM). HPP-HOE140 was radioiodinated using the chloramine-T method (23) and purified on a Sep-Pak C18 cartridge using a 20–80% gradient of methanol diluted in 0.1% trifluoroacetic acid. Nonspecific binding was determined in parallel incubations containing 1 µM unlabeled HOE140. At the completion of the incubation, slides were transferred through four successive 2-min washes in ice-cold 170 mM Tris-HCl buffer (pH 7.4). Competition studies were performed on consecutive sections of rat uterus from ovariectomized and steroid-supplemented animals, using unlabeled bradykinin analogs: the B2 receptor antagonists HOE 140 and D-Arg[Hyp3,D-Phe7,Leu8]bradykinin, the bradykinin B1 receptor antagonist des-Arg9[Leu8]bradykinin, T-kinin, and bradykinin at concentrations ranging from 10-5-10-14 M. At least 15 different concentrations of each unlabeled compound were assessed in duplicate for each treatment. The slides were air-dried and exposed to x-ray film (Agfa Mammoray, Mortsel, Belgium) for 1–2 days. A set of radioactivity standards was prepared by applying known amounts of 125I radioactivity to disks of tissue sections 5 mm in diameter and 10 µm thick, mounted on gelatin-chrom alum-coated slides. These standards were exposed simultaneously with the incubated slides to allow quantification of receptor density by computerized densitometry using an image analysis system (MCID, Ontario, Canada). Across all stages and treatments, the areas of endometrium and myometrium were identified by overlaying the corresponding hematoxylin- and eosin-stained section with the image on the x-ray film. From this, representative areas of the endometrium, which included both stromal and glandular regions, were sampled. Similarly, all layers of the myometrium were included in the myometrial sample, as it was extremely difficult to differentiate between the layers with the image analysis system used. The gray value of the pixels on x-ray film in those areas was converted to disintegrations per min of [125I]radioligand bound/mm2 for quantification of the binding studies. The data from the competition study were analyzed on an iterative model-fitting program using GraphPad Prism (San Diego, CA), where an F test determined the curve of best fit. After exposure to x-ray film, the slides were fixed in 4% paraformaldehyde and dehydrated in increasing concentrations of ethanol before being dipped in liquid emulsion (LM-1, Amersham International) to more precisely define the cellular localization of receptor binding. Slides were exposed for 4–10 days before being developed in D19 developer (Eastman Kodak Co., Rochester, NY), fixed (Ilford Rapidfix, Ilford Imaging Australia, Mt. Waverly, Victoria, Australia), and counterstained with hematoxylin and eosin.

Localization of bradykinin B2 receptor by immunohistochemistry
The B2 receptor polyclonal antibodies (AS276-AS283) were generated against the intra- and extracellular domains of the rat B2 receptor sequence and raised in rabbits. A more detailed description of antibody generation and characterization has been previously reported (24). Immunostaining was performed using the peroxidase-antiperoxidase method with the following modifications. Briefly, 10- or 20-µm frozen sections, taken from the uterine horn of a cycling rat, were mounted on slides, fixed in acetone for 30 sec, washed in PBS (pH 7.6) containing 0.05% Triton, and treated with 100% methanol-1% H2O2 for 20 min followed by incubation with 10% normal goat serum in PBS for 30 min to reduce nonspecific binding. A 1:300 dilution of a pooled combination of eight antipeptide antibodies was used to immunostain for the B2 receptor. Incubation with this antibody combination was carried out in a moist chamber overnight at 22 C. Sections were washed in PBS containing 0.05% Triton before incubation with a 1:200 dilution of antirabbit IgG antibody for 2 h at 22 C. To allow visualization of immunostaining, sections were incubated with avidin-biotin peroxidase complex (Vectastain ABC Kit, Vector Laboratories, Inc., Burlingame, VT) and developed with 3'3-diaminobenzidine and 0.03% H2O2 (SigmaFast DAB tablets, Sigma Chemical Co., St. Louis, MO) for approximately 10 min in the dark. Negative controls for the immunostaining procedure were prepared by omission of the primary antibody or by preabsorbing the AS276-AS283 pooled antipeptide antibodies with an excess of the eight synthetic peptides from the intra- and extracellular domains of the rat bradykinin B2 receptor (30 µg/ml).

Localization of bradykinin B2 receptor messenger RNA (mRNA) by in situ hybridization histochemistry
A PCR fragment of the human bradykinin B2 receptor (nucleotides 541–963; accession no. gi138799) was subcloned in pGEM4Z (Promega Corp., Sydney, Australia). The human and rat (accession no. gi456682) bradykinin B2 receptor nucleotide sequences are 88% identical over this region and therefore cross-hybridize extensively. Sense and antisense RNA transcripts that were 422 bases in length were generated and labeled with [35S]UTP using in vitro transcription (MAXIscript, Ambion, Inc., Austin, TX). The in situ hybridization procedure has been described previously (25) and was followed with minor modifications. Briefly, fresh-frozen sections (10 or 20 µm) were collected onto poly-L-lysine-coated slides at -20 C. Before the hybridization procedure the slides were equilibrated to 22 C, defatted in Histolene (Tronine Pty. Ltd., Riverstone, New South Wales, Australia) and rehydrated in decreasing concentrations of ethanol before being fixed in 4% paraformaldehyde. The prehybridization procedure consisted of rinsing the slides in 0.85% sodium chloride before microwave treatment in 0.01 M citrate buffer (pH 6.0) for 12 min on high. Once the slides cooled, they were postfixed in 4% paraformaldehyde, proteinase K treated (20 µg/ml), and fixed again before dehydration in increasing concentrations of ethanol. 35S-Labeled sense and antisense riboprobes (1 x 106 cpm/ml; ~0.02 ng/µl) were added to a hybridization cocktail consisting of 50% formamide, 10% dextran sulfate, 2 x SSC (standard saline citrate), 70 mM dithiothreitol, and 1 mg/ml denatured salmon sperm. The hybridization mixture was applied to the sections, which were then covered by a Parafilm (American National, Greenwich, CT) coverslip and placed in a humidified chamber for 16 h at 50 C. After hybridization, the coverslips were removed, and sections were washed in a series of buffers at different temperatures before ribonuclease A treatment (20 µg/ml) as described by Sibony et al. (25). Slides were washed further before dehydration through increasing concentrations of ethanol. The air-dried sections were exposed to Amersham ß-Max Hyperfilm (Amersham International) for 21 days. After exposure to x-ray film, the slides were dipped in liquid emulsion (LM-1, Amersham International) for higher power resolution.

Statistical analysis
The differences in B2 receptor binding levels were analyzed using a one-way ANOVA (SigmaStat, Jandel Scientific Software, San Rafael, CA), and Dunnett’s t test was used for post-hoc multiple comparisons. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Localization of the bradykinin B2 receptor by in vitro receptor autoradiography
The radioligand [125I]HPP-HOE140 bound to B2 receptors in the circular and longitudinal smooth muscle layers of the myometrium and to the endometrial stroma across all stages of the rat estrous cycle (Fig. 1Go). Emulsion autoradiography revealed this binding to be localized to the stratum submucosum and stratum vasculare of the myometrium in uteri at diestrus (Fig. 2AGo). In the endometrium at this stage, B2 receptor binding was evident over the glandular epithelium and the layer subjacent to the luminal epithelium and was lower and diffuse throughout the stroma (Fig. 2AGo). Nonspecific binding determined in the presence of 1 µM HOE140 did not produce a detectable image in either the myometrium or endometrium (Fig. 2BGo). Differences in intensity of the B2 receptor at early proestrus and estrus compared with that on diestrus are clearly evident in Fig. 1Go and will be described later. A more specific cellular distribution, as seen on emulsion autoradiography (data not shown), is described below. A similar distribution of B2 receptors was visible in the uterus at early proestrus; however, in this instance, binding in the stratum submucosum of the myometrium and the endometrium was not as intense as distribution in the stratum supravasculare. In the late proestrous stage, binding to the stratum submucosum and stratum vasculare of the myometrium was more intense and extended throughout the stratum supravasculare; however, the stratum suberosum was devoid of binding. The distribution of the B2 receptor in both the myometrium and endometrium at estrus was similar to that seen in the diestrus and early proestrous stages.



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  		Figure 1. Darkfield photomicrographs of the distribution of bradykinin B2 receptors in rat uteri at different stages of the estrous cycle using [125I]HPP-HOE140 as the ligand. The stages are: A, diestrus; B, early proestrus; C, late proestrus; and D, estrus. The white areas represent radioligand binding. E, Endometrium; M, myometrium.

 


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  		Figure 2. Localization of bradykinin B2 receptors in rat uterus at the diestrus stage using [125I]HPP-HOE140 as the radioligand. Sections were stained with hematoxylin and eosin. In A, the black grains depict areas of total binding. B represents nonspecific binding determined in the presence of 1 µM HOE140. G, Gland; L, lumen; S, stroma; smuc, stratum submucosum; sub, stratum suberosum; svas, stratum supravasculare; vas, stratum vasculare.

 
Localization of bradykinin B2 receptors by immunohistochemistry
Histological examination of the uterus sections used for the antibody staining suggested that the rat was at the diestrous stage of the cycle. Positive immunostaining in this tissue was detected in all the strata of the myometrium (Fig. 3AGo), throughout the stroma of the endometrium, as well as over the glandular epithelium and luminal epithelium (Fig. 3BGo). Positive immunoreactivity was also detected over the smooth muscle cells of blood vessels (Fig. 3GGo). Interestingly, preabsorbtion with an excess of peptides eliminated immunostaining in the endometrium (Fig. 3DGo) and smooth muscle cells of the blood vessels (Fig. 3HGo), whereas only a reduction in staining was seen in the stratum suberosum and stratum supravasculare of the myometrium (Fig. 3CGo). The inability of the synthetic peptide mixture to completely block antibody staining in the stratum suberosum and stratum supravasculare may have been due to the low concentration of peptides used. The omission of the antibody produced no detectable staining (Fig. 3Go, E and F).



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  		Figure 3. Immunostaining of bradykinin B2 receptors in rat uterus at the diestrous stage using antibodies generated against synthetic peptides corresponding to the B2 receptor sequence. Sections have been stained with hematoxylin and eosin. The brown areas depict B2 receptor-like immunoreactivity. A, C, and E, Myometrium. B, D, and F, Endometrium. G and H, Blood vessels in the myometrium. A, B, and G, Uterine sections incubated with the mixture of eight different antipeptide antibodies (AS276–AS283) at a dilution of 1:300. C, D, and H, Sections incubated with the mixture of antibodies preabsorbed with an excess of the corresponding eight synthetic peptides (30 µg/ml). E and F, Sections where the primary antibody was omitted from the incubation. bv, Blood vessel; G, glands; L, lumen; S, stroma; smuc, stratum submucosum; sub, stratum suberosum; svas, stratum supravasculare; vas, stratum vasculare.

 
Localization of bradykinin B2 receptor mRNA by in situ hybridization histochemistry
B2 receptor mRNA was localized to the epithelial cells of the endometrial lumen across all stages of the cycle (Fig. 4Go). In contrast, in the myometrium, B2 receptor mRNA was detected in the strata suberosum and supravasculare only at the late proestrous stage (Fig. 4CGo). The sense riboprobe did not produce any visible signal in the in situ experiments (Fig. 4Go, insets a–d).



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  		Figure 4. Darkfield photomicrographs depicting the distribution of bradykinin B2 receptor mRNA in rat uteri at different stages of the estrous cycle: A, diestrus; B, early proestrus; C, late proestrus; and D, estrus. White areas represent B2 receptor mRNA expression. Insets a–d represent nonspecific expression determined by applying the sense riboprobe to the sections.

 
Regulation of the bradykinin B2 receptor throughout the estrous cycle
The B2 receptor levels, as assessed by quantitative in vitro receptor autoradiography, and the serum estradiol and progesterone levels are summarized in Fig. 5Go and Table 1Go, respectively. In the endometrium, a similar density of B2 receptors was detected during the diestrous, late proestrous, and estrous stages, whereas a significantly lower density of receptors occurred at early proestrus (P < 0.05). In the myometrium, a significantly lower density of receptors was detected at the early proestrous stage, compared with that on diestrus, whereas the highest density of receptors occurred during the late proestrous stage. This high density of B2 receptors coincided with the estrogen surge during the late proestrous phase (Fig. 5Go and Table 1Go). These quantitative data confirm the qualitative differences observed in Fig. 1Go.



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  		Figure 5. Bradykinin B2 receptor levels in rat uterus during the different stages of the estrous cycle. i) *, P < 0.05 compared with diestrous group; ii) n represents the number of animals per group; iii) mean ± SEM.

 

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  		Table 1. Serum estradiol and progesterone levels during the different stages of the estrous cycle

 
Regulation of the bradykinin B2 receptor by estrogen and progesterone
The serum estradiol and progesterone levels, summarized in Table 2Go, indicate the effectiveness of ablation and steroid replacement treatments. The greater range and large SE in the sham-operated group were as expected in a random cycling group of animals.


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  		Table 2. Serum estradiol and progesterone levels from ovariectomized/steroid supplemented rats

 
There was an obvious correlation between estradiol levels and increased B2 receptor densities in the myometrium, as demonstrated in the estradiol-supplemented animals compared with control ovariectomized animals (Fig. 6Go and Table 2Go). Two of the four animals in the estradiol-supplemented group that had the highest estradiol levels (180,225 pmol/liter) also had increased bradykinin B2 receptor binding in the endometrium (1,457 dpm/mm2), but for the remaining two animals in this group, which had the lower levels of estradiol (100 pmol/liter), there was not a significant increase in endometrial bradykinin B2 receptor density (385 dpm/mm2). Furthermore, progesterone supplementation alone had no effect on the myometrial or endometrial B2 receptor levels. However, in the estrogen/progesterone-treated animals, progesterone blocked the estrogen induction of myometrial B2 receptors (Fig. 6Go).



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  		Figure 6. Bradykinin B2 receptor densities in rat uteri after ovariectomy with or without steroid supplementation. i) *, P < 0.05 compared with control group; ii) #, P < 0.05 compared with estradiol-supplemented group; iii) n represents the number of animals per group; iv) mean ± SEM.

 
Ligand specificity
The apparent binding affinities of the bradykinin analogs for the bradykinin B2 receptor, as determined by quantitative in vitro receptor autoradiography in ovariectomized rats, are summarized in Table 3Go. These data are graphically represented in Figs. 7Go and 8Go. The bradykinin B1 receptor antagonist, des-Arg9[Leu8]bradykinin, failed to compete for [125I]HPP-HOE140 binding even at concentrations up to 1 µM (data not shown), indicating the binding is specifically to the B2 receptor subtype.


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  		Table 3. Binding affinities (EC50, nanomolar concentrations) unless otherwise stated) for bradykinin analoges in competing for [125I]HPP-HOE140 radioligand from bradykinin B2 receptor receptors on rat uterine sections

 


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  		Figure 7. Competition of [125I]HPP-HOE140 binding to the endometrium of oophorectomized rats supplemented with maize oil (controls; {square}), progesterone ({blacksquare}), estradiol ({circ}), and estradiol/progesterone (X) by D-Arg[Hyp3,D-Phe7,Leu8]bradykinin in A; HOE140 in B, bradykinin in C, and T-kinin in D.

 


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  		Figure 8. Competition of [125I]HPP-HOE140 binding to the myometrium of oophorectomized rats supplemented with maize oil (controls; {square}), progesterone ({blacksquare}), estradiol ({circ}), and estradiol/progesterone (X) by D-Arg[Hyp3,D-Phe7,Leu8]bradykinin in A, HOE140 in B, bradykinin in C, and T-kinin in D.

 
HOE140 elucidated only a single site in both the myometrium and endometrium across all treatment groups, except in the myometrium of estradiol-supplemented animals and the endometrium of control animals (Table 3Go and Figs. 7BGo and 8BGo). In the myometrium and endometrium of ovariectomized rats supplemented with maize oil or progesterone, competition of [125I]HPP-HOE140 by the B2 receptor antagonist, D-Arg[Hyp3,D-Phe7,Leu8]bradykinin (Figs. 7AGo and 8AGo), or bradykinin (Figs. 7CGo and 8CGo) elucidated only a single site. Similarly, in the myometrium and endometrium of the ovariectomized animals supplemented with both estrogen and progesterone, only a single low affinity site was evident by D-Arg[Hyp3,D-Phe7,Leu8]bradykinin (Figs. 7AGo and 8AGo) or bradykinin (Figs. 7CGo and 8CGo) competition. In contrast, in rats supplemented with estradiol, two sites, a low affinity and an additional high affinity site, were observed for all of the peptides competing for [125I]HPP-HOE140 binding in the myometrium. Two sites were also observed in the endometrium of these estradiol-supplemented animals when using bradykinin and D-Arg[Hyp3,D-Phe7,Leu8]-bradykinin as the competing ligands; however, only the low affinity site was evident when HOE140 was used as the competing ligand.

In contrast, T-kinin (Figs. 7DGo and 8DGo) elucidated high and low affinity sites in the myometrium and endometrium of the ovariectomized rats supplemented with maize oil and the rats supplemented with estradiol. In the myometrium of ovariectomized rats supplemented with both estrogen and progesterone, only a single site was elucidated by T-kinin; however, in the endometrium of these rats, T-kinin elucidated both a high and a low affinity site.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The development of selective and potent bradykinin B2 receptor antagonists, such as HOE140, which displays a higher affinity and specificity than previous B2 receptor antagonists (26, 27, 28, 29), has enabled the delineation and characterization of the bradykinin B2 receptor protein by affinity cross-linking studies (30). In this current study, the B2 receptors are localized using [125I]HPP-HOE140, an analog of the specific B2 receptor antagonist. We have previously shown that this radioligand is specific and has a high affinity for the bradykinin B2 receptor (17, 22). In female reproductive organs, bradykinin has been implicated in smooth muscle contraction (7, 13), vasodilation (1), and maintaining uteroplacental blood flow (8, 9); however, less is known about the cellular distribution and regulation of the receptor thought to mediate these actions in the uterus.

Even though B2 receptors are known to be present in myometrial tissues (6, 31, 32, 33), this is the first study to localize B2 receptors at the cellular level in both the myometrium and endometrium, using three independent experimental methods. The B2 receptor protein was detected in the circular and longitudinal smooth muscle layers of the myometrium and in the stroma, glandular epithelium, and the cells adjacent to the lumen of the endometrium using in vitro receptor autoradiography and immunohistochemistry. These findings correlate well with those of Figueroa et al. (20), who detected immunoreactive B2 receptors in the glandular and luminal epithelium and stromal cells of the endometrium and the inner third of the myometrium in rat and human uterus (20). The distribution of the B2 receptor in the rat at diestrous is also consistent with our previous findings in the guinea pig uterus at the same stage (17). The endometrial localization of the B2 receptor in the rat correlates well with the localization of bradykinin-like immunoreactivity in the human endometrium, where staining was detected in the glandular epithelium and stroma (19); however, in that study, the myometrium was not examined. Expression of B2 receptor mRNA was localized to the cells adjacent to the endometrial lumen across all stages of the cycle. In the myometrium, however, B2 receptor mRNA was only detected in the strata suberosum and supravasculare of uteri in the late proestrus stage. In the human uterus, although myometrial expression was not examined, the B2 receptor mRNA was localized to the glands and stromal cells of the endometrium (34). The endometrial stromal B2 receptor mRNA localization contrasts with our findings in the rat uterus, but concurs with our autoradiography and immunolocalization data at the protein level. This may be due to species differences at the mRNA level, or alternatively, this technique may have a decreased sensitivity, although this has not been conclusively proven. In our study, the localization of B2 receptor mRNA in the myometrium of rats during late proestrus, a time when estradiol levels are at their highest, suggests that estrogen regulates myometrial B2 receptor expression. This is contrary to the observations of Madeddu et al. (35), who reported that neither ovariectomy nor estrogen supplementation affected the uterine levels of B2 receptor mRNA (35). However, in that study, the measurements were performed on mRNA extracted from the whole uterus.

We also demonstrate that B2 receptor levels in the myometrium and endometrium are modulated during the estrous cycle, and these changes may be due to regulation by estrogen and progesterone, as myometrial B2 receptor levels are at their highest when estradiol levels peak, and both myometrial and endometrial B2 receptors are at their lowest when progesterone levels peak. Regulation of tissue kallikrein by estrogen and progesterone has also been described. Immunoreactive kallikrein levels were increased during the proestrous phase of the estrous cycle, suggesting that bradykinin levels may be increased at this time (15). Interestingly, immunoreactive kallikrein content in the uterus increased significantly in ovariectomized rats treated with either 0.5 or 5 µg estradiol, whereas progesterone supplementation (5 mg) decreased the uterine immunoreactive kallikrein content (36). Tissue kallikrein gene expression in the human endometrium was also elevated midcycle, suggesting an induction by the rising estrogen levels at this time and a role for kallikrein in the proliferation of the endometrium before implantation (37).

Although the role of bradykinin in myometrial physiology is well established (1, 9, 16), its role in the endometrium is less clear. Bradykinin mobilizes intracellular calcium and induces DNA synthesis in quiescent endometrial stromal cells, suggesting that bradykinin may act as a growth factor in these cells (38). In cultured uterine glandular epithelial cells, bradykinin enhances sodium absorption (39) and increases arachidonic acid release, stimulating PG synthesis from both endometrial stromal cells and glands (40), suggesting a role for bradykinin in maintaining the uterine electrolyte environment and the regulation of menstrual bleeding, respectively. In support of these possible roles for bradykinin, our finding shows positive bradykinin B2 receptor-like immunoreactivity in the glands, stroma, and uterine blood vessels.

In estrogen-primed rats, bradykinin significantly increased the synthesis of PGF2{alpha} and PGE2 from uterine smooth muscle, and this synthesis was completely blocked by a nitric oxide inhibitor, suggesting that the maintenance of bradykinin-induced contractions results from nitric oxide-induced PG synthesis and release (41). It is possible that the differential regulation of the bradykinin B2 receptor, seen both between and within the myometrium and endometrium, is a result of the different functions that these two regions perform and their different relationships with second messenger systems.

In the competition-binding experiments, on rat uterus tissue taken from animals that were ovariectomized and supplemented with steroids, only a single ligand-binding site was elucidated in both the myometrium and endometrium of the control and progesterone-treated groups by bradykinin and the B2 receptor antagonists. Interestingly, in estradiol-treated animals, D-Arg[Hyp3,D-Phe7,Leu8]bradykinin, bradykinin, and HOE140 competition revealed both a high and a low affinity binding site in the myometrium. In the endometrium, the high affinity site was not apparent when HOE140 was the competing ligand; however, D-Arg[Hyp3,D-Phe7,Leu8]bradykinin and bradykinin competition revealed both sites. The appearance of a high affinity site in the estradiol-treated group suggests that the high affinity site is dependent on the presence of estradiol. This is supported by the observation of only a single binding site in the progesterone- and estradiol/progesterone-supplemented animals.

Our findings of both a high and a low affinity site in the myometrium of rats treated with estradiol support the findings of an earlier study using unlabeled bradykinin to compete for [3H]BK binding in rat myometrial membranes, where two binding sites with Ki values of 18 pM and 5.6 nM were identified (6). In contrast, in another study using HOE140 as the competing ligand in a [3H]BK binding assay on myometrial membranes, only a single site with pM affinity (Ki value of 88 pM) was detected (31). Our studies do not provide direct clues as to the nature of the two ligand-binding sites; however, these high and low affinity sites may reflect differential binding states, a high affinity G protein-coupled state, and a low affinity uncoupled state (42).

The competition of [125I]HPP-HOE140 binding by T-kinin demonstrates that T-kinin has a high affinity for B2 receptors and that it may elicit its actions via the B2 receptor. T-kinin has been shown to increase microvascular permeability in rat vascular airways (43) and contract rat uterus (10) with a similar potency to that of bradykinin. However, it had a lower affinity for the bradykinin B2 receptor than bradykinin (10) in rat myometrial membranes. In our study, T-kinin has a similar affinity to bradykinin, but has a lower affinity than HOE140, the parent compound of the radioligand.

The increase in the B2 receptor density observed in animals with a high estradiol level may be due to an increase in the number of receptors, as an increase in myometrial B2 receptor mRNA expression is seen at the late proestrous stage (a time of high estradiol levels). It may also reflect increased stability of the corresponding mRNA and/or an attenuation of the bradykinin B2 receptor down-regulation; however, we have not further addressed these various possibilities.

In summary, we have used three different cellular localization methods (in vitro receptor autoradiography, immunohistochemistry, and in situ hybridization histochemistry) to characterize the cellular distribution of the bradykinin B2 receptor in the rat uterus. The regulation of bradykinin B2 receptors by estrogen and perhaps progesterone, as shown in this study, in conjunction with other studies showing the hormonal regulation of tissue kallikrein (15, 36, 37) and kininogens (13) suggests important roles for the kallikrein-kinin system in both the endometrium and myometrium of the uterus.


   Acknowledgments
 
We thank D. Reeves (Queensland University of Technology, Brisbane, Australia) for assistance with the animal experiments, D. Casley (Department of Medicine, University of Melbourne, Melbourne, Australia) for iodinating the HPP-HOE140 ligand, and G. Ward (Princess Alexandra Hospital, Brisbane, Australia) for performing the steroid hormone assays. We also thank Drs. K. Wirth and B. Schölkens (Hoechst AG, Frankfurt, Germany) for the kind gifts of HOE 140 and HPP-HOE140, and Dr. G. P. Aldred for guidance in synthesizing the riboprobe.


   Footnotes
 
1 This work was supported by grants from the National Health and Medical Research Council of Australia and the Deutsche Forschungsgemeinschaft. Back

Received September 1, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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