Center for Reproduction and Early Life, Institute of Clinical Research, University of Nottingham, Nottingham NG7 2RD, United Kingdom
Submitted 12 May 2004 ; accepted in final form 17 August 2004
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ABSTRACT |
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2-adrenergic receptor; myometrium; potassium channel; preterm labor; uterine contraction
One prominent feature of 2-agonists is their ability to activate K+ channels, leading to cellular hyperpolarization (3, 20). We have shown that the protein levels of both the myometrial
2-AR and the
-subunit of the Ca2+-activated K+ (BKCa) channel decrease by
50% after the onset of labor (7, 8, 21). Anwer et al. (3) demonstrated that isoproterenol, a
-AR agonist, can stimulate BKCa channels in pregnant rat myometrium. In addition, ritodrine, a
2-AR agonist, has also been shown to activate BKCa channels via a G protein and cAMP-dependent phosphorylation cascade in cultures prepared from pregnant human myometrium (12). BKCa channels, which are activated by voltage and increased concentrations of intracellular Ca2+, are abundant in smooth muscle (10, 16, 17) where they play an important role in limiting depolarization, thereby relaxing uterine smooth muscle (2, 15). Although evidence favors phosphorylation as the likely mechanism by which
2-agonist regulation of BKCa channel occurs, little is known regarding the extent of interaction between these two proteins in relation to the mechanisms that determine uterine quiescence and the timing of labor.
We tested the hypothesis that there is a direct association between the 2-AR and BKCa channel that is of physiological and clinical significance during pregnancy and labor. The aim of this study was to explore the interaction between these two proteins in the pregnant human myometrium by investigation of their cellular localization, protein-protein association, and functional correlation with a view to identifying novel signaling cascades as potential therapeutic opportunities for the treatment of preterm labor.
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MATERIALS AND METHODS |
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The myometrial biopsy was taken from the mid-upper margin of the lower uterine incision in women undergoing cesarean section. Women suffering from preeclampsia or other medical conditions, such as diabetes or connective tissue diseases, were excluded from this study. All women were undergoing cesarean section because of previous cesarean section or breech presentation. Samples were collected in physiological salt solution (PSS) for immunofluorescence and isometric tension recording studies and snap-frozen in liquid nitrogen followed by storage at 80°C for Western blotting and immunoprecipitation studies. Each myometrial sample was used once, and only one sample was obtained per patient.
Double-staining immunofluorescence and confocal microscopy. Fresh myometrial tissues were washed two times with 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 intermittent, gentle trituration. The cell suspension was carefully layered on a 60% (vol/vol) Percoll gradient and centrifuged at 22°C, 800 g, for 5 min and then washed two times in HBSS (22°C, 800 g for 5 min each) to remove cell debris. The supernatant was discarded, and cytospin slides were prepared immediately by mixing 100 µl of the pellet (containing cells) with an equal volume of PBS followed by centrifugation at 100 g for 10 min.
Cytocentrifuged myometrial cells were fixed for 20 min in 2% (wt/vol) paraformadehyde and then washed two times with 0.1 M PBS. Cells were subsequently permeabilized with 0.5% Igepal in 0.1 M PBS in a humidity chamber at 4°C for 5 min and then washed with PBS (2 times for 5 min) followed by block with 3% (wt/vol) BSA-1% glycine (wt/vol) in PBS for 15 min at room temperature. Cells were then incubated with both primary antibodies; a mouse monoclonal antibody specific to the 9951113-subunit of the BKCa channel (anti-BKCa
-subunit antibody; Transduction Laboratories) and a polyclonal anti-
2-AR mapping to residues 338413 of the carboxy terminus of the receptor (Santa Cruz Biotechnology) diluted in PBS containing 10% (vol/vol) normal horse serum overnight at 4°C. Cells were subsequently washed, incubated for 30 min in the dark with biotinylated anti-mouse IgG (10 µg/ml; Vector Laboratories, Peterborough, UK), washed, and then incubated with Texas Red-avidin DCS (10 µg/ml Vectorstain Elite; Vector Laboratories) for 30 min followed by further washing. The slides were then incubated for another 30 min with biotinylated anti-rabbit IgG (10 µg/ml; Vector Laboratories) followed by fluorescein-avidin DCS (10 µg/ml Vectorstain Elite; Vector Laboratories) and then washed. Slides were mounted in Vectashield mounting media (Vector Laboratories). Cells were viewed on a Zeiss Axiovert 100 microscope with an LSM 510 confocal scan head (Carl Zeiss, Jena, Germany) and a plan-Apochromat x63 oil immersion objective lens. Images were captured using multitracking with 488-nm argon and 543 HeNe lasers and analyzed using LSM software version 3.2 (Carl Zeiss). Percentage colocalization is based on the fluorescence intensity of the
2-AR signal relative to that of the BKCa
-subunit, after deducting the background intensity.
Immunoprecipitation and Western blotting.
Human myometrium was processed, and immunoprecipitation experiments were performed essentially as described by Matharoo-Ball et al. (22), with the following changes: 1) immunoprecipitation buffer for this study had the following composition: 50 mM Tris·HCl, 150 mM NaCl, 1 mM EDTA, 0.2 mM EGTA, 0.1% SDS, 0.3% sodium deoxycholate, 1:500 protease inhibitor, and 1:100 phosphatase inhibitor, and 2) samples were incubated with antibody-specific IgG agarose beads (according to the species of the immunoprecipitating antibody) at 4°C for 2 h instead of 12 h. The immunoprecipitating antibodies were either anti-BKCa -subunit or anti-
2-AR followed by Western blotting separately (7, 8) with both anti-
2-AR (1:250 dilution) and anti-BKCa
-subunit (1:750) antibodies. Controls were incubated with either mouse or rabbit IgG (DAKO, Glostrup, Denmark) instead of primary antibody. Western blotting was carried out as described previously (7, 8). Immunoblots were then processed (Immun Star; Bio-Rad Laboratories, Hertfordshire, UK) and viewed using an imaging densitometer (ChemiDoc; Bio-Rad). Each experiment was repeated in triplicate.
Isometric tension recording.
Longitudinal myometrial strips (2 x 2 x 10 mm) were mounted under 2 g tension in an organ bath (Letica 01; AD Instruments, Oxfordshire, UK) for isometric tension recording in 10 ml of aerated (95% O2 + 5% CO2) PSS at 37°C. Myometrial contractions were stimulated by 109 M oxytocin (Sigma-Aldrich, Poole, UK). Mechanical responses of myometrial strips were measured by Quadbridge (PowerLab; AD Instruments) and recorded using Chart version 4.2 (PowerLab; AD Instruments). After 1 h of equilibration, cumulative increases of ritodrine (109 to 103 M; Sigma-Aldrich) were applied at 20-min intervals, and the contractile activity was measured during each period.
The effects of the BKCa channel blocker paxilline (Sigma-Aldrich) and 2-AR antagonist ICI-118551 (Tocris Cookson, Bristol, UK) on ritodrine-mediated responses were tested by preincubating myometrial strips for 30 min with either 106 M paxilline or 107 M ICI-118551, followed by cumulative additions of ritodrine. Data are presented as the activity integral calculated during the 20-min period after addition of each ritodrine concentration as a percentage of the control integral obtained for 20 min in oxytocin alone. Concentration-response curves of the activity integral were analyzed by fitting the data to the following equation:
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RESULTS |
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Dispersed myometrial cells in cytospins appeared relaxed and elongated, demonstrating their smooth muscle phenotype. However, because of the hypertrophy myometrial cells undergo during pregnancy, myocytes are often 600 µm long. This has the effect that, even after fixation, the extremities of the cells are often raised above the field of view, imparting a distinct appearance to certain cells (Fig. 1, DF, and Fig. 2, D-F ). The smooth muscle nature of these cells has been confirmed by anti-
-actin staining and electrophysiological characteristics (data not shown). Confocal immunofluorescence showed strong labeling of myometrial
2-ARs. This signal localized to a structure coincident with the BKCa channel in myometrial cells of pregnant labor and nonlabor tissues (Fig. 1, A, C, D, and F). Quantitative double-immunofluorescence studies revealed that 63.7 ± 16.8% of the nonlabor (n = 5)
2-AR signal (Fig. 1B) and 61.7 ± 4% of the labor
2-AR signal (n = 5; Fig. 1E) colocalized with myometrial BKCa channels (P = 0.86). Laser scanning by slicing through the cytosol every 20 µm of both nonlabor (n = 5) and labor (n = 5) provided very little evidence of immunofluorescence intracellularly (Fig. 2).
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DISCUSSION |
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In many smooth muscle tissues, including those of the respiratory tract and vasculature (5, 19), BKCa channels demonstrate relaxant effects. Experimental evidence suggests that 2-AR agonists cause relaxation via G protein-dependent pathways that activate adenylate cyclase and increase cytosolic levels of cAMP, leading to phosphorylation of the channel (11). Recent findings in tracheal smooth muscle have demonstrated that
-adrenergic stimulation can also activate BKCa channels independently of channel phosphorylation via the
-subunit of Gs (18). Our functional studies showing that ritodrine-mediated relaxation is blocked by both paxilline and ICI-118551 to the same extent (maximal relaxation of
50%) suggests that the BKCa channel and
2-AR probably form a regulatory pathway that is prominent in the control of myometrial excitability. It is not possible to conclude from our results whether phosphorylation of the BKCa channel is necessary for the
2-mimetic ritodrine to exert its effects. However, in human cultured myometrial cells, the effect of ritodrine appears to involve both cAMP- and protein kinase A (PKA)-mediated phosphorylation in addition to a direct GTP activation of BKCa channel activity in inside-out patches (12).
The discovery of a signaling complex in rat hippocampal neurones comprising the 2-AR, L-type Ca2+ channels, phosphatase 2A, and adenylate cyclase supports the existence of molecular signaling assemblies that allow for specificity of cellular responses (9). Davare et al. (9) have elegantly demonstrated that addition of the
2-agonist albuterol directly to the patch pipette increased the activity of single L-type Ca2+ channels soon after seal formation of cell-attached recordings. This effect was not observed after bath application of albuterol, suggesting that the close proximity facilitates specificity and rapid interaction of the
2-AR, L-type Ca2+ and key signaling molecules. Moreover, a recent study (21) postulated that
2-AR modulation of membrane excitability occurs, with the latter providing a scaffold that couples BKCa with L-type Ca2+ channels in brain, lung, aortic, and bladder tissues. The human myometrial BKCa channel may also function as part of a similarly complex intracellular network comprising enzymes, scaffolding proteins, and second messenger molecules. In support of this, downstream signaling intermediaries (activated by agonist binding to the
2-AR), such as Gs, PKA, adenylate cyclase, and the cAMP pathway, act directly to modulate myometrial BKCa channel activity. Electrophysiological evidence demonstrates that there is differential coupling between the myometrial BKCa channel and the
-adrenoreceptor, since isoprenaline enhanced macroscopic outward current in myometrial cells of term, pregnant women while reducing outward current in myocytes of nonpregnant myometrium. Outward K+ currents in rat myometrium have also been shown to be influenced in opposing directions by PKA and norepinephrine, depending upon the reproductive status of the animal. Our findings, which demonstrate a significant coexistence of
2-AR and BKCa channels, support the notion that membrane ion channels may be direct targets for the regulatory action of
2-AR in target organs, including human myometrium. This close, intimate association would accelerate and concentrate direct signaling between
2-AR and BKCa channel to achieve relaxation.
The control of human myometrial quiescence during pregnancy and its transformation to a highly contractile state are not fully understood. However, the pivotal role of the BKCa channel and its modulation by an array of chemical mediators identifies it as a key sensor through which many cellular processes may be implemented. These include control of vascular tone, cytokine and hormone secretion, redox processes, and neuronal firing. The 2-AR has a similarly ubiquitous cellular distribution. This suggests that, to execute specific cellular functions in response to a physiological stimulus, subcellular compartmentation must be organized such that coupling between receptors and ion channels is optimized. We provide evidence for a protein-protein interaction between the BKCa channel and
2-AR. The fact that an anti-
2-AR antibody could precipitate BKCa channel protein and vice versa is further evidence to indicate that these two proteins exist in close proximity. The
2-AR has a molecular mass between 56 and 85 kDa (4), with 67 kDa reported as being the mature form of the receptor. In our earlier study, the myometrial
2-AR was reported to have a molecular mass of 67 kDa, whereas immunoprecipitation followed by Western blotting, as described herein, identifies a 52-kDa protein. We propose that the reported size differences may be attributed to the heavily glycosylated or phosphorylated nature of this protein. A 56-kDa
2-AR protein was observed in
2-AR-transfected COS-7 cells along with a 43-kDa deglycosylated form of the receptor (25). Although glycosylation does not interfere with
2-AR function, it is considered to be pivotal in determining the correct delivery of the
2-AR to the cell membrane. It appears that the immunoprecipitation protocol we used preferentially detects the truncated form of the
2-AR, reflecting possible chemical modifications to the parent molecule at a site distinct from the antibody recognition site.
Our results provide compelling evidence in favor of a direct interaction between the 2-AR and BKCa channels as a novel component in the mechanism of uterine relaxation and therefore gestational quiescence. Here we show a close association of this receptor-channel coupling at a molecular and functional level. We did not observe any significant difference in either colocalization or dissociation of the two proteins between nonlabor and labor tissues. It is unclear whether myometrial relaxation and the transition to contractions at term involves a direct and immediate interaction between
2-AR and BKCa channels, an action through second-messenger pathways, or indeed activation of alternative signaling pathways that favor contractility. However, it is interesting to speculate from a clinical perspective that bypassing the
2-AR and targeting BKCa channels in human myometrium instead may, in time, deliver a drug superior in terms of its tocolytic efficacy and apparent absence of desensitization, ultimately alleviating the economic and emotional burden of prematurity.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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