Contribution of coupling between human myometrial {beta}2-adrenoreceptor and the BKCa channel to uterine quiescence

Boonsri Chanrachakul, Fiona Broughton Pipkin, and Raheela N. Khan

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


    ABSTRACT
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The {beta}2-adrenergic receptor ({beta}2-AR) and the large-conductance Ca2+-activated K+ (BKCa) channel have been shown, separately, to be involved in mediating uterine relaxation. Our recent studies reveal that the levels of both {beta}2-AR and BKCa channel proteins in pregnant human myometrium decrease by ~50% after the onset of labor. We present direct evidence in support of a structural and functional association between the {beta}2-AR and the BKCa channel in pregnant human myometrium. Localization of both proteins is predominantly plasmalemmal, with 60% of {beta}2-AR colocalizing with the BKCa channel. Coimmunoprecipitation studies indicate that BKCa and {beta}2-AR are structurally linked by direct protein-protein interactions. Functional correlation was confirmed by experiments of human myometrial contractility in which the BKCa channel blocker, paxilline, significantly antagonized the relaxant effect of the {beta}2-AR agonist ritodrine. These novel findings provide an insight into the coupling between the {beta}2-AR and BKCa channel and may have utility in the application of this signaling cascade for therapeutic potential in the management of preterm labor.

{beta}2-adrenergic receptor; myometrium; potassium channel; preterm labor; uterine contraction


PRETERM DELIVERY, defined as delivery before 37 completed weeks of gestation, is the single leading cause of perinatal morbidity and mortality, particularly in developing countries (1). Despite advances in perinatal care, the incidence of preterm delivery has increased progressively over the past two decades (23). The impact of this high rate extends to health care, the economy, education, society, and family. To date, a lack of detailed information pertaining to the cellular mechanisms that determine uterine excitability has hampered the development of new, effective treatments for preterm labor. Drugs active at the {beta}2-adrenergic receptor ({beta}2-AR) have long been used as tocolytic agents, but their widespread tissue distribution has led to adverse maternal cardiovascular and metabolic effects (6, 14). Furthermore, the tachyphylaxis that occurs in vivo after exposure to {beta}2-AR agonists also compromises their effectiveness (13, 24, 26).

One prominent feature of {beta}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 {beta}2-AR and the {alpha}-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 {beta}-AR agonist, can stimulate BKCa channels in pregnant rat myometrium. In addition, ritodrine, a {beta}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 {beta}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 {beta}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.


    MATERIALS AND METHODS
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 RESULTS
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Subjects and tissue collection. This study was approved by Southern Derbyshire Ethics Committee. The following two groups of women were recruited: 1) singleton term pregnant women (gestational age ≥37 wk) undergoing elective cesarean section before the onset of labor and 2) singleton term pregnant women undergoing emergency cesarean section after spontaneous labor (cervical dilatation >3 cm). Written informed consent was obtained from each participant.

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 {alpha}995–1113-subunit of the BKCa channel (anti-BKCa {alpha}-subunit antibody; Transduction Laboratories) and a polyclonal anti-{beta}2-AR mapping to residues 338–413 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 {beta}2-AR signal relative to that of the BKCa {alpha}-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 {alpha}-subunit or anti-{beta}2-AR followed by Western blotting separately (7, 8) with both anti-{beta}2-AR (1:250 dilution) and anti-BKCa {alpha}-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 10–9 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 (10–9 to 10–3 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 {beta}2-AR antagonist ICI-118551 (Tocris Cookson, Bristol, UK) on ritodrine-mediated responses were tested by preincubating myometrial strips for 30 min with either 10–6 M paxilline or 10–7 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:

where y is the response, ymax is the maximum relaxation achieved, ymin is the minimum relaxation achieved, and X is the drug concentration. Data of activity integral were presented as means ± SE. Significance was determined by F-test and Student's t-test as appropriate. A P value <0.05 was considered statistically significant.


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With the exception of the functional studies, results were compared using myometrium from nonlaboring and laboring women on the basis that parturition may be preceded by a physical dissociation between the {beta}2-AR and the BKCa channel that would be detectable by immunoprecipitation or colocalization studies.

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, D–F, and Fig. 2, D-F ). The smooth muscle nature of these cells has been confirmed by anti-{alpha}-actin staining and electrophysiological characteristics (data not shown). Confocal immunofluorescence showed strong labeling of myometrial {beta}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) {beta}2-AR signal (Fig. 1B) and 61.7 ± 4% of the labor {beta}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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Fig. 1. Double-fluorescence labeling on the plasma membrane of myometrial cells from nonlaboring (A–C) and laboring (D–F) pregnant women. Cells were incubated with selective antibody to {alpha}-subunit Ca2+-activated K+ (BKCa) channels (Texas red; A and D) and {beta}2-adrenergic ({beta}2-AR) receptors (fluorescein; C and F). After superimposition (B and E), the yellow signal demonstrates the colocalization of {beta}2-AR and BKCa channels. Magnification is 630-fold in A–F. The photomicrographs of the 2 cells shown are representative of at least 5 cells.

 


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Fig. 2. Sagittal section of myometrial cells demonstrated double-fluorescence labeling of myometrial cells from nonlaboring (A–C) and laboring (D–F) pregnant women. Cells were incubated with selective antibody to {alpha}-subunit BKCa channels (Texas red; A and D) and {beta}2-AR (fluorescein; C and F). After superimposition (B and E), the yellow signal demonstrates the colocalization of {beta}2-AR and BKCa channels. Magnification is 630-fold in A–F. The photomicrographs of the 2 cells shown are representative of at least 5 cells.

 
In light of the double-immunofluorescence studies, we examined whether {beta}2-AR and BKCa channels were structurally associated. Coimmunoprecipitation experiments were performed with the same antibodies as for the double-staining immunofluorescence studies. Thus myometrial membrane proteins were immunoprecipitated with either anti-{beta}2-AR or anti-{alpha}-BKCa antibody. Both {beta}2-AR (52 kDa) and BKCa {alpha}-subunit (125 kDa) protein bands were demonstrable after immunoblots were probed with either anti-{beta}2 AR or anti-BKCa {alpha}-subunit antibody (Fig. 3). Positive results were demonstrated in proteins from both pregnant women with (n = 5) and without (n = 5) labor. No specific bands were detected when the precipitating antibodies were replaced by rabbit or mouse IgG.



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Fig. 3. Immunoprecipitation of {beta}2-AR and {alpha}-subunit BKCa channels. Myometrial membrane proteins from laboring (L) and nonlaboring (NL) pregnant women were immunoprecipitated (IP) with either anti-{beta}2 AR (A) or anti-{alpha}-subunit BKCa channels (B). Control (C) lanes were immunoprecipitated with mouse or rabbit IgG. Thereafter, Western blots were probed with either anti-{beta}2-AR or anti-{alpha}-subunit BKCa channels. Results are representative of 5 independent experiments with identical results.

 
On the basis of the observed colocalization and coimmunoprecipitation findings, we performed additional isometric tension recordings of nonlabor myometrium to investigate the effect of the specific BKCa channel blocker paxilline on the relaxant effect of the {beta}2-AR agonist ritodrine. The relaxation observed with cumulative additions of ritodrine (n = 15) was antagonized by 1 x 10–6 mol/l paxilline (n = 15), apparent as a rightward shift in the concentration-response curve (Fig. 4). The ritodrine concentration required for 50% maximum response (EC50) was 6 x 10–6 (SE 0.7) mol/l and 3 x 10–4 (SE 0.6) mol/l in the absence and presence of 1 x 10–6 mol/l paxilline, respectively (P < 0.01). The magnitude of the rightward shift observed with paxilline mirrored that obtained with the specific {beta}2-AR antagonist ICI-118551 [EC50 = 0.87 x 10–4 (SE 0.9) mol/l; P < 0.01].



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Fig. 4. Concentration-response curves for the effect of cumulative additions of ritodrine on myometrial contractility (percentage contraction achieved). Response is shown in the absence (control, {blacksquare}) and presence of 10–6 mol/l paxilline ({circ}) and 10–7 mol/l ICI-118551 ({blacktriangleup}). Paxilline and ICI-118551 produce rightward displacements of the curve, with a significant shift in EC50 ({star}P < 0.01) for both. Data of activity integral were presented as the mean ± SE percentage of the results obtained before any drug application for each individual strip. The concentration-response curves were analyzed by fitting to the equation: , where y is the response, ymax is the maximum relaxation achieved, ymin is the minimum relaxation achieved, and X is the drug concentration.

 

    DISCUSSION
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 MATERIALS AND METHODS
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We present herein immunochemical evidence for an apparently direct protein-protein interaction between the human myometrial {beta}2-AR and the BKCa channel. Furthermore, our pharmacological data lend support to the hypothesis that a functional signaling complex formed by these two membrane proteins mediates relaxation in pregnant human myometrium and may constitute a cellular mechanism whereby uterine quiescence is maintained throughout pregnancy. We and others have shown that the BKCa channel, activated by raised intracellular Ca2+ levels, is of significance in maintaining the resting membrane potential of isolated myometrial cells and, by opposing depolarization, would, at a tissue level, prevent uterine contractions (2, 16). The BKCa channel, by integrating multiple inputs from diverse stimuli, influences and affects numerous cellular pathways. However, the mechanisms by which signaling is spatially and specifically routed are not fully understood.

In many smooth muscle tissues, including those of the respiratory tract and vasculature (5, 19), BKCa channels demonstrate relaxant effects. Experimental evidence suggests that {beta}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 {beta}-adrenergic stimulation can also activate BKCa channels independently of channel phosphorylation via the {alpha}-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 {beta}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 {beta}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 {beta}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 {beta}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 {beta}2-AR, L-type Ca2+ and key signaling molecules. Moreover, a recent study (21) postulated that {beta}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 {beta}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 {beta}-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 {beta}2-AR and BKCa channels, support the notion that membrane ion channels may be direct targets for the regulatory action of {beta}2-AR in target organs, including human myometrium. This close, intimate association would accelerate and concentrate direct signaling between {beta}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 {beta}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 {beta}2-AR. The fact that an anti-{beta}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 {beta}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 {beta}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 {beta}2-AR protein was observed in {beta}2-AR-transfected COS-7 cells along with a 43-kDa deglycosylated form of the receptor (25). Although glycosylation does not interfere with {beta}2-AR function, it is considered to be pivotal in determining the correct delivery of the {beta}2-AR to the cell membrane. It appears that the immunoprecipitation protocol we used preferentially detects the truncated form of the {beta}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 {beta}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 {beta}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 {beta}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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B. Chanrachakul was supported by the Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Thailand.


    ACKNOWLEDGMENTS
 
We thank Tim Self for advice on laser scanning confocal microscopy and Anita Turner and Dr. Balwir Matharoo-Ball for assistance with immunoprecipitation studies. Thanks also to the staff and patients in the Department of Obstetrics & Gynecology, Derby City General Hospital, for making this study possible.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. N. Khan, Center for Reproduction and Early Life, Institute of Clinical Research, Univ. Of Nottingham, Academic Division of Obstetrics and Gynaecology, The Medical School, Derby City General Hospital, Uttoxeter New Road, Derby DE22 3DT, UK (E-mail: raheela.khan{at}nottingham.ac.uk)

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
1. American College of Obstetricians and Gynecologists. Assessment of risk factors for preterm birth. Obstet Gynecol 98: 709–716, 2001.[Free Full Text]

2. Anwer K, Oberti C, Perez GJ, Perez-Reyes N, McDougall JK, Monga M, Sanborn BM, Stefani E, and Toro L. Calcium-activated K channels as modulators of human myometrial contractile activity. Am J Physiol Cell Physiol 264: C976–C985, 1993.

3. Anwer K, Toro L, Oberti C, Stefani E, and Sanborn BM. Ca2+-activated K+ channels in pregnant rat myometrium: modulation by a beta-adrenergic agent. Am J Physiol Cell Physiol 263: C1049–C1056, 1992.[Abstract/Free Full Text]

4. Barak LS, Ferguson SS, Zhang J, Matenson C, Meyer T, and Caron MG. Internal trafficking and surface mobility of a functionally intact beta2-adrenergic receptor-green fluorescent protein conjugate. Mol Pharmacol 51: 177–184, 1997.[Abstract/Free Full Text]

5. Barman SA, Zhu S, Han G, and White RE. cAMP activates BKCa channels in pulmonary arterial smooth muscle via cGMP-dependent protein kinase. Am J Physiol Lung Cell Mol Physiol 284: L1004–L1011, 2003.[Abstract/Free Full Text]

6. Bulbring E and Tomito T. Catecholamine action on smooth muscle. Pharmacol Rev 39: 49–96, 1987.[ISI][Medline]

7. Chanrachakul B, Matharoo-Ball B, Turner A, Robinson G, Broughton-Pipkin F, Arulkumaran S, and Khan RN. Immunolocalization and protein expression of the alpha subunit of the large-conductance calcium-activated potassium channel in human myometrium. Reproduction 126: 43–48, 2003.[Abstract/Free Full Text]

8. Chanrachakul B, Matharoo-Ball B, Turner A, Robinson G, Broughton-Pipkin F, Arulkumaran S, and Khan RN. Reduced expression of immunoreactive beta(2)-adrenergic receptor protein in human myometrium with labor. J Clin Endocrinol Metab 88: 4997–5001, 2003.[Abstract/Free Full Text]

9. Davare MA, Avdonin V, Hall DD, Peden EM, Burette A, Weinberg RJ, Horne MC, Hoshi T, and Hell JW. A beta2 adrenergic receptor signaling complex assembled with the Ca2+ channel Cav1.2. Science 293: 98–101, 2001.[Abstract/Free Full Text]

10. Erulkar SD, Ludmer J, Ger B, and Nori RD. Expression of different potassium channels in cells isolated from human myometrium and leiomyomas. Am J Obstet Gynecol 168: 1623–1639, 1993.[ISI][Medline]

11. Gilman AG. G proteins: transducers of receptor-generated signals. Annu Rev Biochem 56: 615–649, 1987.[CrossRef][ISI][Medline]

12. Hamada Y, Nakaya Y, Hamada S, Kamada M, and Aono T. Activation of K channels by ritodrine hydrochloride in yterine smooth muscle cells from pregnant women. Eur J Pharmacol 288: 45–51, 1994.[CrossRef][Medline]

13. Hausdorff WP, Caron MG, and Lefkowitz RJ. Turning off the signal: desensitization of beta-adrenergic receptor function. FASEB J 4: 2881–2889, 1990.[Abstract]

14. Jeyabalan A and Caritis SN. Pharmacologic inhibition of preterm labor. Clin Obstet Gynecol 45: 99–113, 2002.[ISI][Medline]

15. Khan RN, Morrison JJ, Smith SK, and Ashford MLJ. Activation of large conductance potassium channels in the pregnant human myometrium by pinacidil. Am J Obstet Gynecol 178: 1027–1034, 1998.[ISI][Medline]

16. Khan RN, Smith SK, Morrison JJ, and Ashford ML. Properties of large conductance potassium channels in human myometrium during pregnancy and labour. Proc R Soc Lond B Biol Sci 251: 9–15, 1993.[ISI][Medline]

17. Khan RN, Smith SK, Morrison JJ, and Ashford MLJ. Calcium-dependence and pharmacology of large-conductance potassium channels in nonlabour and labour uterine myocytes. Am J Physiol Cell Physiol 273: C1721–C1731, 1997.[Abstract/Free Full Text]

18. Kume H, Graziano MP, and Kotlikoff MI. Stimulatory and inhibitory regulation of calcium-activated potassium channels by guanine nucleotide-binding proteins. Proc Natl Acad Sci USA 89: 11051–11055, 1992.[Abstract]

19. Kume H, Hall IP, Washabau RJ, Takagi K, and Kotlikoff MI. Beta-adrenergic agonists regulate KCa channels in airway smooth muscle by cAMP-dependent and -independent mechanisms. J Clin Invest 93: 371–379, 1994.[ISI][Medline]

20. Kume H, Takagi K, Tokuno H, and Tomita T. Regulation of Ca-dependent K-channel activity in tracheal myocytes by phosphorylation. Nature 341: 152–154, 1989.[CrossRef][ISI][Medline]

21. Liu G, Shi J, Yang L, Cao L, Park SM, Cui J, and Marx SO. Assembly of a Ca(2+)-dependent BK channel signaling complex by binding to beta2 adrenergic receptor. EMBO J 23: 2196–2205, 2004.[Abstract/Free Full Text]

22. Matharoo-Ball B, Ashford ML, Arulkumaran S, and Khan RN. Down-regulation of the alpha- and beta-subunits of the calcium-activated potassium channel in human myometrium with parturition. Biol Reprod 68: 2135–2141, 2003.[Abstract/Free Full Text]

23. Mattison DR, Damus K, Fiore E, Petrini J, and Alter C. Preterm delivery: a public health perspective. Paediatr Perinat Epidemiol 15, Suppl 2: 7–16, 2001.

24. Pitcher JA, Freedman NJ, and Lefkowitz RJ. G protein-coupled receptor kinases. Annu Rev Biochem 67: 653–692, 1998.[CrossRef][ISI][Medline]

25. Rands E, Candelore MR, Cheung AH, Hill WS, Strader CD, Dixon RA. Mutational analysis of beta-adrenergic receptor glycosylation. J Biol Chem 265: 1059–1064, 1990.[Abstract/Free Full Text]

26. Ruzycky AL and DeLoia JA. Expression of beta-adrenergic receptor kinase subtypes in the pregnant rat myometrium. Am J Obstet Gynecol 176: 1077–1083, 1997.[ISI][Medline]