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Pregnancy |
- and ß-Subunits of the Calcium-Activated Potassium Channel in Human Myometrium with Parturition1
Academic Division of Obstetrics & Gynaecology,3 University of Nottingham, Derby City General Hospital, Derby DE22 3NE, United Kingdom
Department of Pharmacology & Neuroscience,4 Ninewells Hospital Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom
Department of Obstetrics & Gynaecology,5 St. George's Hospital Medical School, London SW17 0RE, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and/or ß-subunit or arises from a physical dissociation of the -subunit from the ß-subunit. The ß-subunit is a key determinant of BKCa-channel Ca2+ sensitivity. Western blot analysis, using - and ß-subunit-specific antibodies, detected bands of 110–125 and 36 kDa, respectively. Protein expression levels of the -subunit in term labor myometrium were significantly reduced compared with term pregnancy without labor. Furthermore, -subunit levels at term pregnancy were significantly increased relative to the nonpregnant state, whereas levels at preterm gestations were unchanged. Densitometric analysis demonstrated significantly decreased ß-subunit levels in term and preterm labor samples compared with term nonlabor samples. Immunoprecipitation studies revealed the presence of both the - and ß-subunits in samples taken before or after the onset of labor. We conclude that during labor, the -subunit is not physically uncoupled from the ß-subunit, but a decline occurs in the level of ß-subunit protein, which may underlie the loss of calcium and voltage sensitivity of the BKCa channel with labor. Furthermore, reduced ß-subunit protein in preterm labor myometrium implies that ion channels may also contribute to pathophysiological labor.
calcium, female reproductive tract, parturition, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Uterine myometrial contractility at term is triggered by a number of physiological signals, which orchestrate changes in uterine excitability via ion-channel modulation. The high-conductance, voltage-dependent, calcium-activated potassium channel (BKCa) is the predominant potassium-channel type expressed in nonpregnant [2, 3] and pregnant [4–6] human myometrium. This channel is unique in that it is activated by membrane depolarization and by an increase in the intracellular calcium concentration ([Ca2+]i), thereby playing a pivotal role in modulating uterine function [4, 6, 7].
The smooth muscle BKCa channel is formed by tetrameric assembly of an -subunit and an accessory ß-subunit [8–10]. To date, a family of four mammalian BKCa ß-subunits (ß1 through ß4) has been described [9–14], with each family member having a characteristic tissue distribution and varied effects on BKCa-channel pharmacology and gating. The ß1-subunit consists of two glycosylated transmembrane segments [8, 9], which combine with the -subunit in a 1:1 ratio [8]. Cloning and coexpression studies have revealed that whereas the -subunit forms the functional BKCa channel, coexpression of the ß1-subunit imparts greater Ca2+ and voltage sensitivity [15, 16] in addition to altering the pharmacological responses of the channel [17, 18].
We have previously reported the presence of a potassium channel in human labor myometrium lacking Ca2+ and voltage dependence [5, 6]. This channel, denoted BK to indicate large conductance and Ca2+ independence, also displays an altered pharmacological profile. The molecular mechanisms by which the BKCa channel responds to fluctuating [Ca2+]i remain an enigma, although the following mechanisms have been proposed: 1) tissue-specific expression of the ß-subunit [19], 2) presence of a "Ca2+ bowl" located between the S9 and S10 tail region of the -subunit [20], 3) phosphorylation-dephosphorylation of the -subunit [21], and 4) alternative mRNA splicing of the slo gene, which encodes the BKCa channel, to generate a large number of distinct channel isoforms that differ in their sensitivity to voltage and [Ca2+]i [22–24].
In light of the fact that the ß-subunit is an important determinant of BKCa-channel voltage and Ca2+ sensitivity, we hypothesized that the Ca2+ insensitivity of this channel with labor could arise by a dissociation between the - and ß-subunits of the BKCa-channel complex or by a reduction in protein levels of the ß-subunit. The aim of the present study was to investigate these two possibilities by Western blot analysis of human myometrium at term and preterm gestations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Triton X-100, prestained molecular mass standards, nitrocellulose, and Immunstar chemiluminescent reagents were obtained from Bio-Rad Laboratories (Herts., U.K.). Minicomplete protease inhibitor cocktail tablets were from Boehringer-Mannheim Biochemical (Lewes, U.K.); alkaline phosphatase-conjugated (swine anti-rabbit immunoglobulin [Ig] G and goat anti-mouse IgG) antibodies were obtained from DAKO (Ely, U.K.). Dynabeads were purchased from Dynal (Warrington, U.K.). Anti-ßslo(118–132) and anti-slo(913–926) were a generous gift from Dr. Maria Garcia, Merck Pharmaceutical (Rahway, NJ). All other reagents and chemicals were obtained from Sigma Chemical Company (Poole, U.K.)
Tissue Collection
Myometrial samples were collected from women undergoing either elective or emergency cesarean section at preterm (26–36 wk) or term (38–41 wk) gestations and were divided into the following groups: 1) term nonlabor (TNL), 2) term labor (TL), 3) preterm nonlabor (PTNL), and 4) pretem labor (PTL). Biopsy specimens were taken from the midline of the upper section of the lower uterine segment. The reasons for cesarean section included breech presentation, previous cesarean section, cephalopelvic disproportion, failure of labor to progress, or fetal distress. None of the women included in this study had evidence of underlying disease (e.g., hypertension, diabetes, preeclampsia, intrauterine growth restriction, etc.). As we have previously reported that the presence of the BK (calcium-independent) channel was positively correlated with progressive cervical dilatation [5], we defined labor as cervical dilatation (>4 cm) accompanied by regular uterine contractions. Nonpregnant myometrium (NP) was obtained from normal, cycling patients undergoing hysterectomy for nonmalignant disease. All samples were snap-frozen in liquid nitrogen immediately after collection and stored at -80°C until processing. Written informed consent was obtained from all donors. The study had the approval of the Southern Derbyshire Ethics Committee, England.
Membrane Preparation
Approximately 0.5 g of human myometrial tissue was homogenized in ice-cold buffer containing: 25 mM Tris-HCl (pH 7.4), 300 mM sucrose, 0.25 mM PMSF, 1.0 mM EGTA, 0.1% (v/v) Tween 20, a minicomplete protease inhibitor cocktail tablet, 0.1 mg/ml of benzamidine, and 8 µg/ml each of calpain I and II. After removal of cell debris by centrifugation (1000 x g, 10 min, 4°C), the resulting supernatant was centrifuged (100 000 x g, 60 min, 4°C) to obtain the crude membrane fraction. The latter was resuspended in resuspension buffer: 25 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1.0 mM EGTA, 0.25 mM PMSF, 0.1% (v/v) Tween 20, and a minicomplete protease inhibitor cocktail tablet. For immunoprecipitation studies (see below), the frozen membrane pellets were thawed and solubilized in ice-cold buffer containing 50 mM Tris-HCl (pH 7.4) with 1% (v/v) NP-40 by shaking for 1 h. The solubilized protein was obtained by centrifugation at 14 000 x g for 15 min. Protein determinations were carried out using the bicinchoninic protein assay kit with bovine serum albumin as standard.
Immunoprecipitation Studies
Aliquots (200 µg) of solubilized membrane protein fractions were prepared in a final volume of 200 µl of immunoprecipitation buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% (v/v) NP-40, and a minicomplete tablet. To each sample, one of the following was added: 5 µl of rabbit polyclonal anti-slo(913–926) antibody (directed toward residues 913–926) of the C-terminus of the BKCa-channel -subunit, or 5 µl of the affinity-purified antibody anti-ßslo(118–132) (directed toward residues 118–132 of the ß-subunit of the BKCa channel). Samples were then incubated for 8 h at 4°C under gentle rotation. Next, 30 µl of goat anti-rabbit IgG agarose slurry (1:1 [v/v]) in immunoprecipitation buffer or 30 µl of sheep anti-rabbit IgG Dynabeads M-280 (resuspended in immunoprecipitation buffer) were added to all samples and incubated for 12 h at 4°C with gentle rotation. Immunoprecipitated samples were collected by sedimentation in a microcentrifuge and then washed three times with 500 µl of ice-cold immunoprecipitation buffer. The immunoprecipitated subunits were analyzed by SDS-PAGE after first denaturing the samples for 15 min at 70°C in SDS sample buffer containing 1% (v/v) ß-mercaptoethanol. Sample buffer consisted of the following: 0.125 mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol, and 0.02% (w/v) bromophenol blue.
Western Blot Analysis
Protein samples (100 µg) were heated at 70°C for 15 min after addition of an equal volume of a 2x sample buffer. The membrane fraction and immunoprecipitated proteins were then resolved by electrophoresis on a 10% SDS-PAGE gel at a constant current of 20 mA/gel for 40 min (Protean III; Bio-Rad) with a running buffer containing 0.025 mM Tris (pH 8.3), 0.192 mM glycine, and 0.1% (w/v) SDS. The separated proteins were then transferred to nitrocellulose membrane at a constant voltage of 100 V at 4°C for 60 min. To ensure transfer and equal loading of proteins, blots were stained with Ponceau S solution. Membranes were then blocked for 2 h at room temperature with Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-TE; pH 7.4), and 10% (w/v) low-fat milk powder (Marvel). Blots were then incubated overnight at 4°C with a 1:300 dilution of the affinity-purified rabbit polyclonal antibody anti-slo(913–926) or a 1:4000 dilution of the affinity-purified anti-ßslo(118–132) antibody, diluted in TBS-TE containing 3% (w/v) Marvel (Premier Brand UK, Ltd., Lincolnshire, U.K.). Blots were washed five times with TBS-TE and incubated for 2 h at room temperature with alkaline phosphatase-conjugated swine anti-rabbit IgG secondary antibody diluted in TBS-TE containing 3% (w/v) Marvel. After several washes in TBS-TE, blots were incubated with Immunstar enhanced chemiluminescence reagents and then chemiluminescence detected (Chemidoc hardware; Bio-Rad). Specificity of antibodies was verified by preadsorption experiments in which a 5-fold excess of antibody-specific peptide antigen was preincubated with the appropriate antibody before carrying out Western blot analysis.
Analysis
Protein expression levels of the - and ß-subunits were determined by laser densitometric analysis (Quantity One; Bio-Rad). Peak count values were expressed as densitometric units and mean densitometric units ± SEM for each comparison group. Differences between groups were determined by ANOVA with post-hoc Scheffe analysis using the statistics program SPSS (version 11.0; SPSS, Inc., Chicago, IL). A probability level of P < 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and ß-subunits were investigated by Western blot analysis employing subunit-selective antibodies (anti-slo(913–926) and anti-ßslo(118–132) in uterine membrane fractions prepared from myometria of NP, TNL, PTNL, TL, and PTL women. The BKCa-channel - and ß-subunit proteins were detected in all myometrial tissue membrane fractions examined. In all experiments, transfer and equal loading of 100 µg of protein was confirmed after electroblotting by staining the nitrocellulose membrane with Ponceau S stain. The pattern of protein expression was shown to be reproducible and consistent by carrying out the experiments multiple times using the same and different membrane samples.
-Subunit Expression
Western blot analysis of myometrial membrane fractions demonstrated that anti-slo(913–926) antibody recognized a band of 100–120 kDa in NP (n = 8), TNL (n = 14), PTNL (n = 7), TL (n = 15), and PTL (n = 7) myometrial membranes (Fig. 1A). Figure 1B depicts a scatter plot as well as the mean densitometric units for each experimental group, excluding the NP group. The highest levels of -subunit protein expression were found in the TNL group and the PTNL group (Fig. 1B). On comparing -subunit protein levels between groups (Fig. 1B), a significant decrease was detected between the TL (n = 15) experimental group compared with TNL (n = 14) samples (P < 0.01). No detectable change was apparent when PTL and PTNL -subunit levels were compared with the TNL group (P > 0.05) (Fig. 1B). A marked increase was also observed between TNL (n = 19) levels when compared with the NP (n = 7) group (P < 0.05) (see Fig. 4). However, no difference was observed between -subunit levels of TL (n = 18) compared with NP samples (n = 7; P > 0.05) (Fig. 4). Multiple bands of lower molecular weight than the 110–120 kDa of the BKCa -subunit were detected in all myometrial samples when the membranes were blotted with the BKCachannel subunit anti-slo(913–926) antibody; these bands are protein fragments of the documented proteolytic degradation of the -subunit [25]. The 110- to 120-kDa band is specific for the BKCa channel, because these bands were absent when the anti-slo(913–926) antibody was preincubated, before immunoblotting, with the corresponding peptide antigen against which the antibody was generated (Fig. 2).
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ß-Subunit Expression
The anti-ßslo(118–132) antibody recognized a band corresponding to 36 kDa in myometrial membranes of NP (n = 8), TNL (n = 19), PTNL (n = 8), PTL (n = 8), and TL (n=14) myometrial samples (Fig. 3A). Post-hoc (Scheffe) statistical analysis revealed a significant (P < 0.05) reduction in ß-subunit protein in both the TL (n = 14) and PTL (n = 8) groups compared with the PTNL (n = 8) and TNL (n = 19) groups. The difference in ß-subunit levels was also significant (P < 0.01) between TL (n = 17) and TNL (n = 22) samples (Figs. 3 and 4). The most dramatic difference was evident as a reduction in ß-subunit levels in TL (n = 17) and TNL (n = 22) compared with NP (n = 8) tissue (P < 0.01) (Fig. 4). The specificity of anti-ßslo(118–132) antibody for the ß-subunit was shown by the absence of ß-signal in blots probed after preadsorption of anti-ßslo(118–132) with its corresponding antigen (data not shown).
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Immunoprecipitation Studies
To ascertain whether the loss of Ca2+ sensitivity of this channel with labor may be caused by an uncoupling of the ß1-subunit from the -subunit, we investigated anti-slo(913–926) and anti-ßslo(118–132) immunoprecipitation of the BKCa-channel /ß-subunit complex from myometrial samples. Immunoprecipitation with either anti-slo(913–926) and anti-ßslo(118–132) followed by probing with anti-slo(913–926) unveiled a 110-kDa band (Fig. 5A). Conversely, immunoprecipitation with anti-slo(913–926) (Fig. 5B) and anti-ßslo(118–132) (data not shown) and blotting with anti-ßslo(118–132) produced a 36-kDa band corresponding to the ß-subunit of the BKCa channel in both these groups. This suggests a coupling of the BKCa-channel /ß-subunit complex in both the TL and TNL groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-actin in human TL myometrium (unpublished results); 2) enzymatic dispersal of postlabor myometrium, which predominantly yields myocytes that have been used for electrophysiological studies [5, 6]; and 3) high levels of smooth muscle -actin in crude membrane fractions of TL myometrium detected by Western blot analysis with anti--actin antibody (data not shown). Furthermore, isometric tension recordings of human myometrial strips from the upper and lower segment failed to reveal any differences in contractile properties [26]. However, differences in expression levels for a variety of proteins including connexin-43, cyclooxygenase (COX)-1, COX-2, [27], and splicing factors SF2/ASF [28] have been observed between upper-segment and lower-segment myometrial biopsy specimens as well as with labor and in the nonpregnant state. Those authors [28] have also reported similar expression levels of U2-B''snRNP protein in nonpregnant samples in addition to upper- and lower-segment biopsy specimens of nonlabor and labor myometrium. The unique physiological and anatomical adaptations with pregnancy and labor within the myometrium indicate that generalizations regarding function and protein expression cannot be drawn and must be interpreted in light of these dynamic changes. Because the present study did not utilize upper-segment myometrial biopsies, it is not known whether BKCa-channel protein expression displays spatial differentiation between fundal and corporal tissue sampling sites. In the present study, Western blot analysis of human myometrium from the lower segment, obtained at defined gestational stages, was performed to investigate the protein expression of the BKCa - and ß-subunits to establish if either subunit has a role in the onset of human labor.
The size (110–120 kDa) of the main protein band detected by the anti-slo(913–926) antibody used in the present study is consistent with that reported for Western blot analysis of the rat [29], mouse [30], and human [31] myometrial BKCa channel as well as the bovine tracheal [25] and rat brain -subunit [32]. Our results demonstrate that expression levels of the two BKCa-channel subunits do vary during pregnancy and with labor. Previously, it has been reported that levels of the -subunit in TNL human myometrium were unchanged in comparison to NP tissue, but labor myometrium was not investigated as part of that study [31]. Our finding that -subunit levels are significantly elevated during pregnancy compared with the NP state contrasts with those results [31] and also with findings in rat myometrium [29], in which a 60% decline in BKCa -subunit protein levels close to or at term was noted relative to nonpregnant tissue. An increase in immunoreactive BKCa -subunit protein at term compared with the nonpregnant state has been reported in mouse myometrium [30]. In the present study, stringent criteria were adopted for obtaining labor myometrium at the time of cesarean section. The fact that -subunit levels were dramatically reduced postpartum in the animal studies [29, 30] supports the idea of reduced -subunit levels following the onset of labor compared with the TNL state, as described here. Because the -subunit is the pore-forming unit of the channel itself and the BKCa channel is particularly abundant in uterine myocytes, a reduction of approximately 40% during term labor (as obtained in the present study) in protein levels of this subunit would translate into a considerable shift toward electrical excitability in the human uterus and likely promote contractile activity. Conversely, it appears that raised BKCa-channel levels during pregnancy relative to parturition provide an effective hyperpolarizing current directed at maintaining an inexcitable uterus.
The significant diminution reported herein for the BKCa ß-subunit of 50% and 75% following PTL and TL, respectively, relative to the TNL state is far greater than that found with the -subunit. A 45% reduction in ß-subunit levels in TNL tissue compared with NP myometrium was also observed. This is close to the 34% reported by Zhou et al. [31] for a similar comparison in human myometrium, leading those investigators to consider it unlikely that the ß-subunit could contribute to the higher BKCa-channel activity observed during pregnancy. Why levels of the ß-subunit are higher in NP myometrium than during pregnancy is unclear, but these elevated levels may be related to Ca2+-dependent physiological mechanisms, whereby small shifts in intracellular Ca2+ levels during the menstrual cycle and/or implantation could be important for cellular function. Interestingly, the highest levels of -subunit protein (TNL group) did not correlate with high levels of ß-subunit protein (NP group).
We also investigated the possibility that the BKCa channel is uncoupled from its accessory ß-subunit during labor. Immunoprecipitation studies using anti-slo(913–926) and anti-ßslo(118–132) demonstrated that both - and ß-subunits were detectable when immunoblotted with the appropriate antibodies, as reported by Knaus et al. [9, 10]. The fact that anti-slo(913–926) antibody detected the main 110-kDa band in myometrial samples immunoprecipitated with anti-ßslo(118–132) antibody suggests that the close association between these two subunits is still intact during gestation and that the reported loss of Ca2+ dependence of the BKCa channel is not simply caused by a physical separation between the two subunits. Rather, the transition to a Ca2+-independent K+ channel at labor may result from the dramatic reduction in ß-subunit levels. Alternative mechanisms whereby changes in Ca2+ and voltage sensitivity of the BKCa channel may reflect tailoring to cellular function include the expression of splice variants of the slo gene, as reported for the electrical tuning of cochlear hair cells [24, 33]. In the pregnant mouse myometrium, multiple BKCa isoforms are differentially expressed and regulated throughout gestation, with the most prominent channel isoform being characterized by a diminished Ca2+ and voltage sensitivity closer to term [33]. Recently, Korovkina et al. [34] described a novel 132-base pair exon of the BKCa channel from pregnant human myometrium, which imparts reduced Ca2+ and voltage sensitivity when expressed in fibroblasts.
It is intriguing to consider how the greatly reduced levels of ß-subunit protein influence the function and biophysical properties of the labor BKCa channel. The BKCa - and ß-subunits were initially shown to exist in a basic 1:1 stoichiometry to form a tetrameric (4:4) channel [8]. Wang et al. [35] recently provided evidence that BKCa channels with a reduced stoichiometry of :ß do assemble, but that the biophysical features of the channel, such as gating behavior and inactivation, are a function of the average number of ß-subunits per channel, with an increase in the ß: ratio imparting greater Ca2+ and voltage sensitivity. The substantial decline in ß-subunit levels reported here for myometrium, initially during pregnancy and a further decrease following labor, would imply that subunit stoichiometry is considerably less than 1:1. Consequently, the Ca2+ dependence of the channel may be greatly compromised and the interaction between the two subunits altered to such an extent that the BKCa channel is rendered Ca2+ insensitive.
Single-channel recordings have unequivocally demonstrated the presence of myometrial BKCa channels [3–5]. However, controversy continues over remodeling of whole-cell Ca2+-sensitive K+ currents in term myometrium and the contribution from the BKCa conductance. This may, in part, be explained by the reduced BKCa ß-subunit levels noted at term in the present study, which would lead to an altered current phenotype that manifests as a Ca2+-independent current component. We propose that the down-regulation of - and ß-subunit proteins in term labor myometrium switches the uterus from a state of relative quiescence throughout pregnancy to its highly contractile state at parturition. This dual, temporally related decline in - and ß-subunit expression may be sufficiently low to bring about a change in the channel's Ca2+ and voltage sensitivity, thereby facilitating myometrial contractility and delivery of the fetus. If such a decline in ß-subunit levels were to occur too early, then this could potentially evoke premature activation of the myometrium, terminating in preterm labor. This is, in fact, borne out by our finding that down-regulation of the ß-subunit occurs with preterm labor, indicating that ion channels may contribute to this pathophysiology. This is, to our knowledge, the first report documenting a potential role for ion-channel subunits in precipitating premature labor. These results also provide a molecular basis for our earlier observation [5, 6] that at term labor, the observed loss of BKCa Ca2+ and voltage sensitivity is a result of drastically reduced ß-subunit protein expression. The identification of the cellular/molecular triggers that bring about changes in both BKCa subunits is clearly necessary to understand the nature of the relationship between ion channels and other contraction-associated proteins (e.g., gap junctions, prostaglandins) in bringing about labor. In conclusion, we propose that the BKCa ß-subunit may be a novel target through which future therapeutic interventions for labor suppression and/or induction may be possible.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Raheela N. Khan, Academic Division of Obstetrics & Gynaecology, Clinical Sciences Building, University of Nottingham, Derby City General Hospital, Derby DE22 3NE, United Kingdom. FAX: 1332 625634; raheela.khan{at}nottingham.ac.uk
Received: 19 August 2002.
First decision: 9 September 2002.
Accepted: 14 January 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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proteins in the upper and lower segments of the human uterus during pregnancy and labor. J Clin Endocrinol Metab 2000; 84:1705–1710 subunit in myometrium. FEBS Lett 1999 460:427-432[CrossRef][Medline] and auxiliary ß subunits on functional properties of large conductance Ca2+-activated K+ channels. J Neurosci 2002 22:1550-1561This article has been cited by other articles:
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  P. J. Brunton, M. Sausbier, G. Wietzorrek, U. Sausbier, H.-G. Knaus, J. A. Russell, P. Ruth, and M. J. Shipston Hypothalamic-Pituitary-Adrenal Axis Hyporesponsiveness to Restraint Stress in Mice Deficient for Large-Conductance Calcium- and Voltage-Activated Potassium (BK) Channels Endocrinology, November 1, 2007; 148(11): 5496 - 5506. [Abstract] [Full Text] [PDF]
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 J. Sharkey and J. Olcese Transcriptional Inhibition of Oxytocin Receptor Expression in Human Myometrial Cells by Melatonin Involves Protein Kinase C Signaling J. Clin. Endocrinol. Metab., October 1, 2007; 92(10): 4015 - 4019. [Abstract] [Full Text] [PDF]
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 A. Shmygol, K. Noble, and S. Wray Depletion of membrane cholesterol eliminates the Ca2+-activated component of outward potassium current and decreases membrane capacitance in rat uterine myocytes J. Physiol., June 1, 2007; 581(2): 445 - 456. [Abstract] [Full Text] [PDF]
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 K. Y. Yoo, J. C. Lee, M. H. Yoon, M.-H. Shin, S. J. Kim, Y. H. Kim, T. B. Song, and J. Lee The effects of volatile anesthetics on spontaneous contractility of isolated human pregnant uterine muscle: a comparison among sevoflurane, desflurane, isoflurane, and halothane. Anesth. Analg., August 1, 2006; 103(2): 443 - 7, table of contents. [Abstract] [Full Text] [PDF]
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 M. Wareing, X. Bai, F. Seghier, C. M. Turner, S. L. Greenwood, P. N. Baker, M. J. Taggart, and G. K. Fyfe Expression and function of potassium channels in the human placental vasculature Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2006; 291(2): R437 - R446. [Abstract] [Full Text] [PDF]
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 C. R. Rosenfeld, T. Roy, K. DeSpain, and B. E. Cox Large-Conductance Ca2+-Dependent K+ Channels Regulate Basal Uteroplacental Blood Flow in Ovine Pregnancy Reproductive Sciences, September 1, 2005; 12(6): 402 - 408. [Abstract] [PDF]
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 A. M. Brainard, A. J. Miller, J. R. Martens, and S. K. England Maxi-K channels localize to caveolae in human myometrium: a role for an actin-channel-caveolin complex in the regulation of myometrial smooth muscle K+ current Am J Physiol Cell Physiol, July 1, 2005; 289(1): C49 - C57. [Abstract] [Full Text] [PDF]
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 T. Suzuki and K. Takimoto Differential expression of Kv4 pore-forming and KChIP auxiliary subunits in rat uterus during pregnancy Am J Physiol Endocrinol Metab, February 1, 2005; 288(2): E335 - E341. [Abstract] [Full Text] [PDF]
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B. Chanrachakul, F. B. Pipkin, and R. N. Khan Contribution of coupling between human myometrial {beta}2-adrenoreceptor and the BKCa channel to uterine quiescence Am J Physiol Cell Physiol, December 1, 2004; 287(6): C1747 - C1752. [Abstract] [Full Text] [PDF]
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P < 0.01 between TL and TNL. For the ß-subunit: 
P < 0.01 when TL and TNL levels were compared with NP and also for TL with TNL group
