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Academic Division of Obstetrics and Gynecology, Derby City General Hospital, University of Nottingham (B.C., B.M.-B., A.T., R.N.K.), Derby, United Kingdom DE22 3NE; Academic Division of Obstetrics and Gynecology, Queen’s Medical Center, University of Nottingham (G.R., F.B.-P.), Nottingham, United Kingdom NG7 2UH; and Academic Division of Obstetrics and Gynecology, St. George’s Hospital Medical School, University of London (S.A.), London, United Kingdom SW17 0RE
Address all correspondence and requests for reprints to: Dr. Raheela N. Khan, Academic Division of Obstetrics and Gynecology, University of Nottingham, Clinical Sciences Building, Derby City General Hospital, Uttoxeter New Road, Derby, United Kingdom DE22 3NE. E-mail: raheela.khan{at}nottingham.ac.uk.
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Abstract |
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Introduction |
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The ß2-adrenergic receptor (ß2-AR), a member of the superfamily of G protein-coupled receptors (4), has a widespread tissue distribution and is expressed abundantly in the smooth muscle of the trachea, bronchi, vasculature, and uterus (5). This receptor plays an important role in smooth muscle relaxation resulting from the activation of the adenylate cyclase signaling cascade. ß2-Agonists have been used as the main tocolytic drug of choice in the treatment of preterm labor for the past 2 decades (5). However, ß2- mimetic therapy for preterm labor is associated with reduced tocolytic efficacy due to the development of tachyphylaxis, resulting from either a reduction of ß2-AR number or ß2-AR uncoupling mainly by the action of G protein-coupled receptor kinase (6, 7, 8). Although there is clearly a clinical need for the development of safe, novel, and more effective tocolytic agents to treat preterm labor, improved understanding of the cellular and molecular mechanisms that underlie the action of ß2-ARs and the subsequent development of tachyphylaxis in the human uterus is needed.
Considering that ß2-agonists have been used as the main treatment for preterm labor for some time, it is surprising that only a few studies have investigated the expression of myometrial ß2-ARs. Obtaining such important fundamental knowledge would aid our understanding of the mechanisms that regulate uterine contractions. Data on the expression of myometrial ß2-AR in animals are inconclusive (9, 10, 11). Legrand et al. (11) reported that the ß2-AR concentration in pregnant rat myometrium is constant and decreases just 6 h before delivery. However, Engstrom et al. (9) showed that ß2-AR number in pregnant rats decreases linearly throughout the gestational period. Only a few studies have been reported regarding the expression of ß2-ARs in human myometrium, with conflicting results. Evidence from radioligand binding studies indicates that levels of total myometrial ß-AR expression remain unchanged during human pregnancy and after the onset of labor, whether term or preterm (12). In contrast, Breuiller et al. (13) demonstrated decreased levels of ß-AR in term, nonlaboring human myometrium. To date, no immunolocalization studies have examined the distribution of ß2-ARs in human myometrium.
In view of these discrepant findings and as part of a wider study into the signaling pathways in the human myometrium during pregnancy and labor, we have used immunochemical methods to compare protein expression and localization of immunoreactive ß2-AR protein in the myometrium of nonpregnant, term nonlaboring, and term laboring women to establish whether temporal changes in ß2-AR expression accompany gestation and the onset of labor.
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Subjects and Methods |
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This study was approved by the Southern Derbyshire ethics committee. Written informed consent was obtained from each participant. Three groups of women were recruited for this study: 1) 8 nonpregnant, menstruating women undergoing hysterectomy for benign disease; 2) 9 term pregnant women (gestational age, 
37 wk) carrying a single fetus and undergoing elective cesarean section before the onset of labor; and 3) 10 similar term pregnant women undergoing emergency cesarean section after spontaneous labor (all women were in the active phase of labor with cervical dilatation 3 cm). The indication for emergency cesarean section was failure to progress in labor. Pregnant women diagnosed as suffering from diabetes mellitus or preeclampsia were excluded from this study.
Myometrial biopsies were taken from the mid-upper margin of the lower uterine segment incision. Biopsies from the lower part of the corpus were taken from nonpregnant women undergoing hysterectomy. For the following experiments, the myometrium was immediately separated from decidua and serosa and divided into three section for immunohistochemistry, immunofluorescence, and Western blotting studies. Polyclonal ß2-AR antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) specific to amino acids 338–413 at the carboxyl terminal of ß2-AR was used as a primary antibody for all experiments.
Immunohistochemistry
Myometrial tissues were immediately fixed in 10% (vol/vol) formal saline. After routine tissue processing by dehydration through an ascending alcohol series, clearing in xylene, and infiltration with paraffin wax, the tissues were embedded, and sections (4 µm thick) were cut on a microtome and collected on 3-aminopropyltriethoxysilane-coated slides. Immunohistochemistry was performed by the avidin-biotin complex technique. Paraffin sections were deparaffinized in xylene and rehydrated in a graded alcohol series for 5 min each. Antigen retrieval was routinely performed by heating slides in a microwave oven for 15 min in citrate buffer (10 nM, pH 6). After this, endogenous peroxidase was blocked by incubation in 0.6% (vol/vol) hydrogen peroxide (H2O2) in methanol. Nonspecific background staining was blocked by nonimmune goat serum. After blotting of excess serum, tissue sections were incubated with a rabbit primary polyclonal antibody to the human ß2-AR (Santa Cruz Biotechnology, Inc.) at a dilution of 1:200 overnight at 4 C, followed by 1 h at room temperature. Negative controls were provided by incubating sections with rabbit IgG (DAKO, Glostrup, Denmark) instead of primary antibody or omitting the primary antibody. Sections were subsequently washed and incubated with biotin-conjugated goat antirabbit secondary antibody (Vector Laboratories, Inc., Peterborough, UK), followed by peroxidase-conjugated avidin biotin complex (Vectastain Elite, Vector Laboratories, Inc.). Positive staining was visualized using 3,3'-diaminobenzidine tetrahydrochloride (Vector Laboratories, Inc.), and the sections were counterstained with Harris hematoxylin (Nustain, Nottingham, UK). The slides were subsequently dehydrated in a graded alcohol series, cleared in xylene, and mounted in DPX (DePex, BDH Chemicals, Poole, UK). Sections were viewed with a DMRB light microscope (Leica, Deerfield, IL), and photographs were taken at appropriate magnifications using an attached modular photomicrographic system (Leica Wild MPS48).
Immunofluorescence
Myometrial tissues were washed twice in HEPES-buffered Ca2+- and Mg2+-free Hanks’ balanced salt solution (HBSS), minced finely in collagenase A in HBSS (2 mg/ml), and incubated in this enzymatic solution for 1 h at 37 C with gentle trituration every 30 min. The cell suspension was carefully layered onto a 60% (vol/vol) Percoll gradient, centrifuged at 22 C at 800 x g for 5 min, and then washed twice in HBSS in readiness for cytospin.
A cytospin of isolated myometrial cells was fixed in 2% paraformaldehyde, permeabilized with 0.5% Igepal in 0.1 M PBS, and then blocked with 3% (wt/vol) BSA-1% glycine (wt/vol) in PBS. Cells were incubated with rabbit primary polyclonal antibody to the human ß2-AR (Santa Cruz Biotechnology, Inc.) at a dilution of 1:50 overnight at 4 C and were subsequently washed and incubated with biotinylated antirabbit IgG (10 µg/ml; Vector Laboratories, Inc.), followed by Texas Red Avidin DCS (10 µg/ ml; Vector Laboratories, Inc.). The slides were mounted in Vectashield mounting medium (Vector Laboratories, Inc.). Cell were viewed with a fluorescence microscope (Axiovert S 100, Carl Zeiss, New York, NY).
Western blotting
Three hundred milligrams of snap-frozen myometrial tissue were homogenized (Heidolph DIAX 900 homogenizer, Heidolph Instruments, Schwabach, Germany) in homogenization buffer [25 mM Tris-HCl (pH 7.4), 300 mM sucrose, 0.25 mM phenylmethylsulfonylfluoride, 1.0 mM EGTA, and 0.1% (vol/vol) Tween 20]. After an initial centrifugation at 1,000 x g for 10 min at 4 C, the supernatant was removed, and a further centrifugation was performed at 100,000 x g for 1 h at 4 C. The resulting pellet was resuspended in homogenization buffer, centrifuged at 1,000 x g for 10 min at 4 C, and total protein content was determined by the bicinchoninic acid method. Eighty micrograms of crude membrane protein were loaded and resolved by electrophoresis on 10% SDS-PAGE gels at 20-mA constant current for 50 min. Proteins were then electroblotted onto nitrocellulose paper. Equal loading protein and transfer were confirmed by Ponceau S staining. Blots were subsequently blocked with 0.1% (vol/vol) Tween in Tris-buffered saline containing 5% (wt/vol) nonfat dry milk (Marvel, Premier Brands Ltd., Lincolnshire, UK) and subsequently incubated at a 1:250 dilution of primary ß2-AR polyclonal antibody in 3% (wt/vol) nonfat milk overnight at 4 C. After washing, the blots were incubated with alkaline phosphatase-conjugated antirabbit IgG at a dilution of 1:1,000 at room temperature for 2 h. The blots were subsequently incubated with Immun-STAR AP reagent (Bio-Rad Laboratories, Hertfordshire, UK). Protein bands were detected and quantified by chemiluminescence (Quantity One software, Bio-Rad Laboratories). Negative controls were provided by sample buffer. Rat brain membrane, prepared with the same method as myometrial membrane, was used as an internal standard and positive control. Forty micrograms of rat brain membrane sample, which gave the best signal for ß2-AR, were loaded on each gel. Rat brain tissue is rich in ß2-AR (14) and therefore provides a suitable reference against which myometrial ß2-AR expression can be compared.
Statistical analysis
Protein expression of the ß2-AR of myometrial membranes was normalized with respect to the rat brain signal (which served as an internal standard). Values were expressed as a percentage relative to the ß2-AR signal intensity in rat brain tissue. Data were checked for normal distribution by Kolmogorov-Smirnov test. All data are expressed as the mean ± SEM. Differences between groups were analyzed by one-way ANOVA with Bonferroni post hoc analysis or two-tailed unpaired t test. Statistical analysis was performed using the statistics program SPSS for Windows version 11.0 (SPSS, Inc., Evanston, IL). The null hypothesis was rejected at a two-tailed P < 0.05.
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Results |
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Figure 1
demonstrates immunoreactive ß2-AR protein in human myometrial sections from nonpregnant and term pregnant women with or without labor. The myometrium of nonpregnant women is composed of closely packed myocytes with oval and spindle-shaped nuclei (Fig. 1, A and D). There is very little cytoplasm and extracellular matrix (Fig. 1, A and D). Myometrial cells from pregnant women are also spindle-shaped and larger in size, but in contrast to cells of nonpregnant women, the former contain a larger amount of cytoplasm (Fig. 1, B–E). The cells are also separated by edema of extracellular matrix (Fig. 1, B–E).
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, A–C). The expression of immunoreactive ß2-AR was visible at the plasma membrane and in the cytosol of the myometrial cells from nonpregnant (Fig. 1D), nonlabor (Fig. 1E), and labor (Fig. 1F) tissues. Positive staining was also observed in smooth muscle cells lining the blood vessels (Fig. 1, D–F).
Immunofluorescence was used to examine the localization of ß2-AR in myometrial cells in more detail. The representative fluorescence images were taken from separated myometrial cells from nonpregnant (Fig. 2A), nonlabor (Fig. 2B), and labor (Fig. 2C) myometria. These results confirmed the immunohistochemical findings. The positive staining observed with Texas Red demonstrates that the distribution of ß2-AR is predominantly expressed at the plasma membrane of nonpregnant, nonlabor, and labor myocytes.
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Myometria from nonpregnant (n = 8), nonlaboring (n = 9), and laboring (n = 10) women was analyzed for the expression of ß2-AR protein. Western blotting of myometrial membranes clearly revealed a 67-kDa band corresponding to ß2-AR in nonpregnant, nonlabor, and labor myometria as well as rat brain membranes (Fig. 3). Duplet bands were demonstrated in some samples (Fig. 3). This might be the result of different phosphorylation states of ß2-AR. Semiquantitative analysis demonstrated that the expression of the myometrial ß2-AR was 70.7 ± 13.9% (n = 8), 62.3 ± 10.5% (n = 9), and 32.8 ± 6.5% (n = 10) in the nonpregnant, nonlabor, and labor states, respectively, compared with rat brain. One-way ANOVA demonstrated a significant difference between groups (P < 0.05). Specifically, the level of ß2-AR protein expression in nonpregnant tissue was significantly higher than that in pregnant women with labor using the Bonferroni test (P < 0.05). Further experiments, designed to have 80% power based on our earlier findings, comparing nonlabor (n = 19) and labor (n = 19) ß2-AR protein levels, revealed that labor was associated with significantly reduced expression levels (23.8 ± 3.9%) compared with the nonlabor state (46.6 ± 8.1%; P < 0.05).
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Discussion |
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, an increase in cAMP, followed by activation of protein kinase A, and consequent myometrial relaxation. The presence and physiological function of ß2-ARs has been clearly documented in the human myometrium (12, 16, 17). However, direct immunolocalization and immunochemical quantification of expression of the human myometrial form of the ß2-AR from the nonpregnant to the parturient state has not been reported. Our immunohistochemical findings demonstrate the presence of immunoreactive ß2-AR protein in myometrial cells of nonpregnant and term pregnant (with and without labor) women. Localization of ß2-AR by immunohistochemistry demonstrated that immunoreactivity to the plasma membrane and cytosol is consistent with findings in human lung fibroblasts and vascular smooth muscle (18). Immunofluorescence also confirmed that the distribution of ß2-AR is predominantly on the plasma membrane of myometrial cells. The cytosolic location of the myometrial ß2-AR is due to normal internalization and trafficking processes that recycle and degrade ß-ARs from the plasma membrane (19, 20).
Immunoblot analysis revealed significantly higher (
2-fold) levels of ß2-AR protein in the nonpregnant and nonlabor myometria compared with the labor myometrium. Contractility of the nonpregnant myometrium directs sperm toward the fallopian tubes for fertilization, facilitates embryo implantation, and expels menstrual effluent (21). However, this activity is weak and, with the exception of menstrual pain, often not experienced by women. The higher ß2-AR levels reported here for myometrium from nonpregnant and pregnant nonlaboring women may therefore contribute to the maintenance of uterine quiescence in contrast to the intense, phasic myometrial activity during labor.
Norwitz et al. (22) described a four-stage sequence during which the myometrium cycles through quiescence, stimulation, activation, and involution during pregnancy and parturition. This series results from a shift in the expression of relaxation-associated proteins toward contraction-associated proteins with labor (23). Our observation that ß2-AR protein levels are significantly reduced after the onset of labor would support a shift away from relaxation-associated proteins with labor. There is little information available regarding ß2-AR expression in human labor myometrium, although unchanged (12) or reduced (13) total ß-AR levels in human myometrium at term compared with preterm gestations have been demonstrated. Further, reduced ß2-AR levels in myometrium of women with preterm contractions after ß2-agonist treatment have been reported (24, 25). It has been estimated that between 65–87% (12, 13, 26) of myometrial ß-AR are of the ß2-subtype, thereby indicating an appreciable ß2-AR component to the measured response. However, a considerable degree of interindividual variation in human myometrial ß2-AR number has been reported (16, 27), and it is therefore conceivable that the reduced responsiveness of individual uteri to ß2-agonist administration may actually lie with an intrinsically low ß2-AR density.
There are other mechanisms involving ß2-AR pathways that could lead to the stimulation of myometrial activity. For example, it has been suggested that it is the uncoupling of the ß2-AR from the adenylate cyclase signaling pathway (16, 28, 29, 30), rather than a specific down-regulation of the ß-AR in the myometrium, that causes desensitization of the ß2-AR response. Furthermore, it has been shown that estrogen and progesterone, key pregnancy hormones, maintain the rat myometrial ß2-AR in a high affinity state (10, 31), whereas dexamethasone is able to reverse the process of ß2-AR desensitization (32).
The debate over whether the physiological triggers for preterm labor are identical to those that lead to term labor remains open. Thus, it is not clear whether the ß2-AR desensitization that occurs at term in the human myometrium (29) is also a feature of preterm labor. Further studies are needed to examine whether the observed reduction of ß2-AR levels in human labor myometrium compared with its nonpregnant and nonlabor counterparts reflects reduced ß2-AR expression concomitantly with the onset of labor or whether this diminution occurs as a consequence downstream of labor. Regardless of the unresolved nature of this temporal association, down-regulation of ß2-AR protein may represent one of several possible mechanisms acting in an orchestrated manner to initiate uterine contractions, thereby triggering the onset of normal or preterm labor. Moreover, our observation of approximately 50% greater ß2-AR levels in nonpregnant and nonlabor human myometria compared with laboring tissue would suggest that knowledge regarding the biology of this receptor in the uterus is complex and incomplete.
In conclusion, diminished ß2-AR levels along with the apparent desensitization of the ß2-AR at term gestation would tend to promote myometrial contractility, thereby initiating labor, probably in concert with other mechanisms. Although the administration of ß2-agonists to suppress preterm labor has its limitations with respect to their safety and efficacy, few novel candidates have emerged as better alternatives. Deeper insight into the molecular mechanisms that govern myometrial ß2-AR expression and desensitization may eventually identify new approaches to maximize the benefits of ß2-AR-mediated tocolysis for the treatment of preterm labor.
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Acknowledgments |
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Footnotes |
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Abbreviations: ß2-AR, ß2-Adrenergic receptor; HBSS, Hanks’ balanced salt solution.
Received April 23, 2003.
Accepted July 2, 2003.
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