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Am J Physiol Heart Circ Physiol 289: H1417-H1427, 2005. First published May 27, 2005; doi:10.1152/ajpheart.01174.2004
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Estrogen regulates {beta}1-subunit expression in Ca2+-activated K+ channels in arteries from reproductive tissues

Deepa Nagar, Xiao-tie Liu, and Charles R. Rosenfeld

Department of Pediatrics, Division of Neonatal-Perinatal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

Submitted 22 November 2004 ; accepted in final form 13 May 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Daily estradiol-17{beta} (E2{beta}) increases basal uterine blood flow (UBF) and enhances acute E2{beta}-mediated increases in UBF in ovariectomized nonpregnant ewes. The acute E2{beta}-mediated rise in UBF involves vascular smooth muscle (VSM) large-conductance Ca2+-activated K+ channels (BKCa). BKCa consist of pore-forming {alpha}-subunits and regulatory {beta}1-subunits that modulate channel function and E2{beta} responsiveness. It is unclear whether E2{beta} also alters subunit expression and thus channel density and/or function, thereby contributing to the rise in basal UBF and enhanced UBF responses that follow daily E2{beta}. Therefore, we examined BKCa subunit expression by using reverse transcription-PCR and immunoblot analysis of arterial VSM from reproductive and nonreproductive tissues and myometrium from ovariectomized nonpregnant ewes after daily E2{beta} (1 µg/kg iv) or vehicle without or with acute E2{beta} (1 µg/kg). Tissue distribution was determined by immunohistochemistry. Acute E2{beta} did not alter {alpha}- or {beta}1-subunit expression in any tissue (P > 0.1). Daily E2{beta} also did not affect {alpha}-subunit mRNA or protein in any tissue (P > 0.1) or mesenteric arterial VSM {beta}1-subunit. However, daily E2{beta} increased uterine and mammary arterial VSM {beta}1-subunit mRNA by 32% and 83% (P < 0.05), uterine VSM protein by 30%, and myometrial {beta}1-subunit mRNA and protein by 74% (P ≤ 0.005). Immunostaining of uterine arteries, myometrium, and intramyometrial arteries paralleled immunoblot analyses for both subunits. Although BKCa density is unaffected by daily and acute E2{beta}, daily E2{beta} increases {beta}1-subunit in proximal and distal uterine arterial VSM. Thus prolonged E2{beta} exposure may alter BKCa function, estrogen responsiveness, and basal vascular tone and reactivity in reproductive arteries by modifying {alpha}:{beta}1 stoichiometry.

myometrium; nonpregnant ewes; vasodilation; uterine blood flow; mesenteric artery; mammary artery

ESTROGEN, A POTENT VASODILATOR, increases blood flow to a variety of tissues in nonpregnant, pregnant, and postpartum animals; however, its greatest effects occur in reproductive tissues, especially the nonpregnant uterus (27, 44, 49). In ovariectomized nonpregnant ewes, daily estradiol-17{beta} (E2{beta}) increases basal uterine blood flow (UBF) by 30–40% after 6–7 days in the absence of changes in arterial pressure or heart rate as well as vascular responsiveness to acute E2{beta} exposure (41, 50), the latter resulting in a greater than fivefold increase in UBF within 90–120 min after an intravenous dose of 1 µg/kg or an arterial dose of 2–4 µg (19, 25, 49). There also are attenuated systemic pressor responses to vasoconstrictors after E2{beta} exposure (31, 39). The effects of E2{beta} on the uterine vasculature are associated with increases in uterine venous plasma and arterial vascular smooth muscle (VSM) levels of cGMP as well as increases in types 1 and 3 nitric oxide (NO) synthase (NOS) in uterine artery VSM and endothelium, respectively (41, 43, 50, 54). Furthermore, the acute rise in UBF and cGMP production after a systemic dose of E2{beta} is inhibited dose dependently up to 70% by the nonspecific NOS inhibitor nitro-L-arginine methyl ester (43, 56). Thus E2{beta}-mediated increases in vascular NO contribute to the rise in UBF and may facilitate attenuated systemic responses to {alpha}-stimulation.

Although NO contributes to E2{beta}-induced vasodilation through increases in vascular cGMP (43, 50), the cellular and molecular pathways resulting in vasodilation are incompletely understood. Darkow et al. (12) observed that acute E2{beta} exposure increased the opening potential (Po) of large-conductance Ca2+-dependent K+ channels (BKCa) in porcine coronary artery myocytes. They subsequently proposed that E2{beta} relaxed coronary arteries by increasing BKCa Po via a cGMP-dependent mechanism that was endothelium independent (59). Rosenfeld et al. (46) also detected BKCa expression in uterine artery myocytes and observed that like the coronary myocyte, acute E2{beta} exposure increased BKCa Po more than 70-fold through an endothelium-independent, cGMP-dependent mechanism. Furthermore, tetraethylammonium chloride, a BKCa-specific inhibitor at submillimolar concentrations (4, 33), dose dependently inhibited the acute E2{beta}-induced rise in UBF when infused into the uterine arterial circulation of ovariectomized nonpregnant ewes (46). Thus E2{beta}-induced uterine vasodilation in nonpregnant ewes involves increases in vascular endothelial NOS (eNOS) and neuronal NOS (nNOS) expression and/or activity that enhance arterial VSM cGMP synthesis, phosphorylate BKCa and increase BKCa Po. Notably, BKCa activation also contributes to E2{beta}-mediated increases in uteroplacental blood flow and to regulation of basal UBF in pregnant ewes (40, 42).

The BKCa consist of four {alpha}-subunits that form the channel pore and one or more isoforms of the {beta}-subunit, which modify BKCa activity and function (10, 18, 22). Although several variants of the {beta}-subunit have been described, the {beta}1-subunit appears to be specific to smooth muscle (34, 35). Increases in {beta}1-subunit expression are associated with enhanced BKCa activity and sensitivity to changes in intracellular calcium, providing a mechanism whereby smooth muscle function and channel phenotype can be altered to adapt to homeostatic changes (9, 11, 21, 37). Levels of {alpha}- and {beta}1-subunit are increased in uterine smooth muscle, i.e., myometrium, from pregnant women and mice, which is believed to enhance uterine quiescence during pregnancy (2, 3, 7, 28, 51). Notably, {beta}1 knockout mice have increases in vascular tone and systemic hypertension (5), and spontaneously hypertensive rats have decreases in VSM {beta}1-subunit (1). The mechanisms regulating these changes in BKCa subunit expression in VSM and myometrium are unclear.

In ovariectomized mice, prolonged E2{beta} exposure increases myometrial {beta}1-subunit mRNA, whereas acute E2{beta} exposure has no effect (3). Thus estrogen may modulate BKCa activity by regulating subunit expression and channel density. Rosenfeld and colleagues (42, 46) have shown BKCa involvement in E2{beta}-mediated uterine vasodilation; thus the enhanced vascular responses and changes in vascular reactivity after daily E2{beta} may reflect changes in BKCa subunit expression and {alpha}:{beta}1-subunit stoichiometry (52, 55). Furthermore, the differences in vascular responses to E2{beta} in reproductive and nonreproductive tissues (44) may also reflect differences in BKCa phenotype. Therefore, we performed studies in chronically instrumented ovariectomized nonpregnant ewes to determine whether acute and/or daily E2{beta} exposure modifies BKCa {alpha}- and {beta}1-subunit expression in VSM from reproductive (uterine and mammary) and nonreproductive (mesenteric) arteries. We also examined the myometrium to determine whether similar changes occurred in nonvascular smooth muscle from the reproductive tract and intramyometrial resistance arteries.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. Tissues were available from 26 nonpregnant ewes of mixed Western breed that were used in studies designed to examine the mechanisms associated with estrogen responsiveness (41, 50). Surgical preparation has been described in detail (19, 41, 50). Briefly, animals were fasted overnight but allowed free access to water. The next morning, animals received atropine sulfate intramuscularly (0.88 µg/kg), and a percutaneous jugular venous catheter was placed for intravenous infusion of preanesthetic agents, sodium pentobarbital (10 mg/kg) and ketamine hydrochloride (1 mg/kg). Animals were intubated, and sterile surgery was performed under inhalation anesthesia with isoflurane (Mallinckrodt Veterinary, Mundelein, IL). Electromagnetic flow probes (3.0–3.5 mm ID; Carolina Medical, King, NC) were implanted around the middle uterine artery of each uterine horn proximal to the first bifurcation. Animals were ovariectomized, and polyvinyl catheters containing heparinized saline (250 U/ml) were implanted into a femoral artery and vein to the level of the descending aorta and abdominal vena cava, respectively. Postoperatively, animals received intravenous flunixin (Banamine, 30 mg; Schering-Plough Animal Health, Union, NJ) for pain and antibiotics for 3 days. Animals were maintained in large individual stalls within the laboratory until the termination of each experiment. Studies were not begun until postoperative day 4 or 5. This animal model was chosen for study because UBF can be continuously monitored, permitting assurance of optimal uterine and systemic responses to the agent being studied, e.g., E2{beta}, and the collection of tissues at times that correlate with physiological responses, e.g., the E2{beta}-mediated increases in UBF (19, 41, 49). The studies described were approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center at Dallas.

Experimental protocols. E2{beta} (Steraloids, Wilton, NH) was dissolved in 95% ethanol and stored at 4°C as a stock concentration of 100 µg/ml, which was allowed to reach room temperature before infusion. Starting on postoperative day 4 or 5, each animal received E2{beta} (1 µg/kg iv) over a 1- to 2-min period each morning while UBF, mean arterial pressure, and heart rate were continuously monitored, beginning 30 min before E2{beta} infusion and continuing for 120 min afterward. This dose of E2{beta} elicits a greater than fivefold rise in UBF by 90–120 min in the unstressed state (19, 25, 49). After establishment of consecutive maximal and reproducible UBF responses to E2{beta} for 2–3 days, animals were randomly placed into four groups. In groups 1 (n = 9) and 2 (n = 8), we sought to compare the effects of vehicle (group 1) and daily E2{beta} (group 2; 1 µg/kg iv) for 6 days on tissues responses. Tissues were collected from these animals on the morning of day 7 to remove any effects of acute E2{beta} exposure. Groups 3 (n = 5) and 4 (n = 4) permitted us to examine the responses to acute E2{beta} exposure after either vehicle or daily E2{beta} for 6 days, respectively. On day 7, both groups received a bolus dose of vehicle or E2{beta} (1 µg/kg iv), and tissues were collected at the time of the maximum rise in UBF (~115 min). Tissue samples were collected as described in Tissue preparation.

Tissue preparation. Animals were killed with an intravenous dose of pentobarbital sodium (120 mg/kg). In addition to collecting the uterine and mesenteric arteries, we also collected samples of the mammary artery because the mammary gland is sensitive to the vascular effects of E2{beta} and demonstrates increases in blood flow after acute and prolonged E2{beta} exposure (19, 27, 44). After both primary mammary arteries were removed, the abdomen was opened and the intact uterus removed in block. A sample of the myometrium was then taken from the uterine body, and first- through fourth-generation uterine arteries were dissected from both uterine horns and placed in sterile chilled physiological saline solution. A large segment of mesenteric artery was then dissected from the omentum and placed in chilled physiological saline solution. While on ice, the adventitia and intraluminal blood were removed from all arterial segments, and samples of each artery were opened along the long axis, the endothelium removed with a soft cotton swab as described previously, and the samples were frozen in liquid nitrogen (41, 50). After the adventitia and endometrium were removed with sharp dissection, the myometrium was cut into 0.3- x 2-cm segments, and the samples frozen in liquid nitrogen. All samples were stored at –80°C until the time of assay. Additional uterine artery and myometrial samples were prepared for immunohistochemistry as described in Immunohistochemistry. Because tissues from animals in groups 1 and 2 had been included in earlier studies (41, 50), tissue samples were not available for each site from each animal, resulting in the difference in the n values noted in RESULTS.

Reverse transcription-PCR. To determine BKCa {alpha}- and {beta}1-subunit expression and regulation in arterial VSM and myometrium, a semiquantitative reverse transcription-PCR (RT-PCR) assay was used (41). Denuded arterial samples and myometrium were removed from –80°C storage, thawed in TRIzol reagent (Invitrogen Life Technologies, Gaithersburg, MD), and homogenized; total cellular RNA was extracted and resuspended in 20 µl of 0.1% diethyl pyrocarbonate water. The concentration and purity were measured at 260/280 nm OD. RT was performed with 2 µg total RNA as previously described (41). The reaction was incubated at room temperature for 10 min and at 37°C for 1 h and was then terminated at 95°C for 5 min. PCR was performed on 2.5 µl of RT product (cDNA) with specific primers designed from nucleotide sequences retrieved from the GenBank (synthesized by Invitrogen Life Technologies). Malate dehydrogenase (MDH) was used as the reference gene to perform semiquantitative RT-PCR. Because the reference gene must not be altered by the experimental paradigm, we initially compared the levels of MDH mRNA in each of the study groups. There was no effect of daily or acute E2{beta} on MDH transcripts in either arterial VSM or myometrium (P > 0.1 by ANOVA); therefore, values for BKCa {alpha}- and {beta}1-subunit mRNA were normalized to MDH, permitting a semiquantitative measure of changes in subunit mRNA.

The primers for MDH were forward 5'-TCCCAGCAGCAACGG-GTGT-3' and reverse 5'-AAATCTTCGGGGTGACAACC-3'. The primers for the BKCa {alpha}-subunit were forward 5'-CAGCATTTGCCGTCAGTGTCCT-3' and reverse 5'-CATGCCTTTGGGTTATTT-TTCC-3'. The primers for the {beta}1-subunit were forward 5'-TCACCTACTACATCCTGGTCACGA-3' and reverse 5'-GGAATTTGGCTCTGACCTTCTCCA-3'. Experimental conditions for the PCR reaction for both the {alpha}- and {beta}1-subunits were optimized. The cycles for DNA amplification were on the linear portion of the assay curve for each artery and run at optimal temperature. The reaction conditions were as follows: 2.5 µl of RT product were added to 2.5 µl of 10x PCR buffer (250 mM Tris·HCl, pH 8.4, 500 mM KCl); 0.5 µl of 10 nM 2-deoxynucleoside 5'-triphosphate; 0.75 µl of 50 mM MgCl2; 0.25 µl of platinum Taq DNA polymerase (Invitrogen Life Technologies); 0.75 µl each of forward and reverse BKCa primers; and 17 µl of distilled water, to make a total of 25 µl of reaction solution. For the {alpha}-subunits, DNA amplification was carried out in 36 sequential cycles at 94°C for 45 s, 62°C for 1 min, 72°C for 1 min, and followed by 72°C for the final extension for 7 min. The {beta}1-subunit required 35 sequential cycles for DNA amplification at 94°C for 45 s, 58°C for 1 min, 72°C for 1 min, followed by 72°C for the final extension for 7 min. For MDH, DNA amplification occurred in 33 sequential cycles at 94°C for 45 s, 58°C for 1 min, 72°C for 1 min, followed by 72°C for the final extension for 7 min. PCR products were size fractionated by applying 5 µl of PCR product to 1.5% agarose gel containing 25 µg/µl ethidium bromide and then visualized under ultraviolet light. Optical densities of DNA bands were scanned and quantified (Scion Image software; Scion, Frederick, MD). When the values were compared, the targeted PCR products were always run on the same gel.

Western blot analysis. Samples of endothelium-denuded uterine, mammary, and mesenteric arteries and myometrium (30 mg) were weighed and homogenized in 40x volumes of SDS buffer as previously described (41). Homogenates were centrifuged at 10,000 g for 2 min. The supernatant was removed, and an aliquot was used to determine cellular or soluble protein by bicinchoninic acid reagent (Pierce, Rockford, IL). Bromophenol blue and 2-mercaptoethanol were added to the aliquots, and equal amounts of soluble protein were then loaded (20 µg) onto 7.5% polyacrylamide minigels and subjected to SDS-polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred to nitrocellulose paper at 100 V for 1 h. The blots were incubated overnight with antisera to BKCa {alpha}-subunit (1:300; {alpha}1098–1196, Chemicon International, Temecula, CA) or the {beta}1-subunit (1:500; Calbiochem, San Diego, CA). After 1 h of incubation with anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (1:2,000), immunoreactive protein was visualized by chemiluminescence. Samples were compared by scanning densitometry with arbitrary units. Ovine cerebellum was used as the positive internal control, and nonimmune rabbit serum was used to show specificity of the antibodies used.

There have been questions regarding the molecular weight of the BKCa {alpha}-subunit and the presence of proteolytic fragments or splice variants (16, 20, 22). Therefore, we performed preadsorption studies with the uterine arterial VSM, myometrium, cerebellum, and the peptide ({alpha}1098–1196) provided with the {alpha}-subunit antibody. The predominant protein band identified in the myometrium was at 83 kDa with a minor band at 105 kDa (Fig. 1A) ; both were absent after preadsorption (Fig. 1B). The uterine arterial VSM demonstrated the same bands, and both were absent after preadsorption (Fig. 1, A and B). In contrast, a single band at ~105 kDa was identified in homogenates of ovine cerebellum (Fig. 1A), and this, too, was absent after preadsorption (Fig. 1B). Importantly, immunostaining for the BKCa {alpha}-subunit peptide ({alpha}1098–1196) at 39 kDa was also absent after preadsorption (Fig. 1, C and D). Thus the ovine cerebellum expresses a protein similar to that in the human and mouse myometrium (2, 7, 28). In subsequent analyses, we used the predominant 83-kDa band to examine changes in subunit protein. Only one protein band was seen for the {beta}1-subunit at 37 kDa, consistent with other reports (2, 16, 22, 28).



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  		Fig. 1. Preadsorption studies of vascular smooth muscle (VSM) cell Ca2+-activated K+ channel (BKCa) {alpha}-subunit antibody to determine protein-antibody specificity, molecular weight, and presence of {alpha}-subunit fragments in myometrium, uterine artery smooth muscle, and ovine brain by using a fivefold excess of competing peptide ({alpha}1098–1196). Each lane was loaded with 20 µg of supernatant or soluble protein (see METHODS). Lane representation is as follows: lanes 1 and 2, myometrium; lanes 3 and 4, uterine artery smooth muscle; lane 5, ovine brain 2 µg soluble protein. A: representative Western immunoblot analysis without preadsorption. B: Western immunoblot analysis after preadsorption with competing peptide. C: {alpha}-subunit control peptide without preadsorption. D: {alpha}-subunit control peptide with preadsorption.

 
Immunohistochemistry. At the time of tissue collection, intact segments of second- and third-generation uterine arteries and myometrium were washed in PBS, fixed in 4% paraformaldehyde for 6 h at room temperature, and embedded in paraffin as previously described (41, 50). Sections were mounted on slides, deparaffinized, hydrated, incubated with avidin-biotin blocking agent for 30 min, and then incubated overnight at room temperature with polyclonal antibodies to the BKCa {alpha}- or {beta}1-subunits (1:30). After endogenous peroxidases were quenched with 3% H2O2 in H2O for 30 min, immunostaining was detected with standard strepavidin-biotin-horseradish peroxidase and hematoxylin counterstaining.

Statistical analysis. Differences between the control and E2{beta}-treated groups were determined using Student's nonpaired t-test. Differences among more than two groups were analyzed by ANOVA for multiple groups with the use of Tukey's test. Data are presented as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Physiological responses to daily E2{beta}. Animals were E2{beta}-free until postoperative day 4 or 5. At that time, each animal received daily E2{beta} (1 µg/kg iv) until UBF responses were maximum and reproducible for 3 days. The animals were then randomly assigned to treatment groups as described in METHODS. Basal mean arterial pressure and heart rate were unaffected by daily E2{beta} throughout the study period. In contrast, basal UBF rose by 36% after 4 days of E2{beta}, increasing from 25 ± 6 to 34 ± 9 ml/min (P < 0.01). Although mean arterial pressure also was unaffected by the bolus doses of E2{beta}, heart rate rose by ~20% after each dose of E2{beta} (P < 0.02) and returned to baseline within 24 h. However, the acute rise in UBF was 43% greater on day 4 of E2{beta} versus day 1 (139 ± 18 vs. 97 ± 14 ml/min; P < 0.01). Thus daily E2{beta} increases basal UBF and is associated with potentiation of the acute rise in UBF after a bolus dose of E2{beta}.

Reproductive arteries. Samples of uterine arterial VSM were available for analysis from several animals within each treatment group described in METHODS, and BKCa {alpha}-subunit transcripts were detected in each sample assayed. There was no difference in the BKCa {alpha}-subunit-to-MDH ratios in the tissues available from animals in the four study groups (Fig. 2 ; P = 0.5 by one-way ANOVA). Thus there was no evidence of an effect of either daily or acute E2{beta} treatment on levels of uterine arterial VSM {alpha}-subunit mRNA. This permitted us to combine the results from groups 1 (vehicle only) and 3 (vehicle plus acute E2{beta}) and groups 2 (daily E2{beta}) and 4 (daily plus acute E2{beta}) to further compare the effects of vehicle (n = 9) and daily E2{beta} (n = 10). Again, there was no significant effect of daily E2{beta} on the BKCa {alpha}-subunit (1.27 ± 0.14 and 1.42 ± 0.15; P = 0.5), respectively. We then examined the protein expression of the BKCa {alpha}-subunit in uterine arterial VSM, performing Western blot analysis with endothelium-denuded arteries from groups 1 and 2. The anti-{alpha}-subunit antibody recognized a single band at 83 kDa in all uterine arterial VSM samples, and as with RT-PCR, there was no effect of daily E2{beta} on BKCa {alpha}-subunit protein contents (Fig. 3 ; P = 0.5).



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  		Fig. 2. Results of reverse transcription-PCR (RT-PCR) for BKCa {alpha}-subunit mRNA in uterine arterial VSM from ovariectomized nonpregnant ewes after treatment with vehicle (n = 4), daily estradiol-17{beta} (E2{beta}; 1 µg/kg; n = 6), vehicle plus acute E2{beta} (1 µg/kg; n = 5), or daily E2{beta} plus acute E2{beta} (n = 4). A: results of RT-PCR for {alpha}-subunit and malate dehydrogenase (MDH). B: results of densitometry in arbitrary units corrected for MDH mRNA. Values are means ± SE. Groups are not different for {alpha}-subunit. P = 0.5 by ANOVA.

 


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  		Fig. 3. Immunoblot analysis of BKCa {alpha}- and {beta}1-subunit protein in uterine arterial VSM from ovariectomized nonpregnant ewes after treatment with vehicle (n = 5) and daily E2{beta} (1 µg/kg; n = 5). A: immunoblot analysis for subunit protein. B: analysis of densitometry in arbitrary units. Values are means ± SE. There is no difference between groups for {alpha}-subunit protein (P = 0.5). In contrast, {beta}1-subunit protein was modestly increased. *P = 0.096 after daily E2{beta}.

 
Transcripts for the BKCa {beta}1-subunit were also present in each of the uterine arterial VSM samples assayed for the {alpha}-subunit. There was no difference between vehicle alone and vehicle plus acute E2{beta} or between daily E2{beta} and daily plus acute E2{beta} (Fig. 4 ; P > 0.3 by ANOVA), demonstrating that acute E2{beta} exposure did not modify {beta}1-subunit mRNA. However, compared with vehicle, {beta}1-subunit mRNA increased >30% after daily E2{beta} or daily plus acute E2{beta} (Fig. 4; P < 0.05 by ANOVA). Because there was no acute effect of E2{beta} on {beta}1-subunit transcripts, we combined the groups as noted above. Thus there was a 40% increase in uterine arterial VSM {beta}1-subunit transcripts after daily E2{beta} (P < 0.001). With the use of Western blot analysis, BKCa {beta}1-subunit protein was detected at 37 kDa in all VSM samples from groups 1 and 2; furthermore, {beta}1-subunit protein contents increased 30% after daily E2{beta}, paralleling the rise in mRNA (Fig. 3; P = 0.096).



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  		Fig. 4. Results of RT-PCR for BKCa {beta}1-subunit mRNA in uterine arterial VSM from ovariectomized nonpregnant ewes after treatment with vehicle (n = 4), daily E2{beta} (1 µg/kg; n = 6), vehicle plus acute E2{beta} (1 µg/kg; n = 5), or daily plus acute E2{beta} (n = 4). A: results of RT-PCR for {beta}-subunit and MDH. B: results of densitometry in arbitrary units corrected for MDH mRNA. Values are means ± SE. Levels of {beta}1-subunit mRNA in the two vehicle groups do not differ from each other (P = 0.5 by ANOVA), and there also is no difference between daily E2{beta} groups. However, values for daily E2{beta} groups are significantly greater than their respective vehicle groups. *P < 0.001 by ANOVA.

 
Although we were primarily interested in the effects of E2{beta} on uterine arterial VSM, we examined the mammary artery as another E2{beta}-sensitive tissue to determine whether similar changes occurred in BKCa subunit expression in VSM from both reproductive arteries. There was no difference in arterial VSM BKCa {alpha}-subunit mRNA or protein between vehicle-treated (n = 4) and daily E2{beta}-treated (n = 5) groups (P > 0.1); however, transcripts of the {beta}1-subunit increased 83% from 0.86 ± 0.2 to 1.54 ± 0.2 (P = 0.02). In contrast to uterine arterial VSM, we did not detect a change in {beta}1-subunit protein in mammary arteries after using Western blot analysis (P > 0.1; data not shown), which was repeated several times.

Nonreproductive artery. Vasodilatory responses to E2{beta} in nonreproductive tissues are less than those seen in reproductive tissues, and the mechanisms for this difference are unclear. It is possible that differences exist in BKCa expression. However, there are no studies of the effects of E2{beta} on BKCa subunit expression in VSM from nonreproductive arteries. Transcripts for both BKCa subunits were present in all of the mesenteric arterial VSM assayed (Fig. 5). In contrast to the uterine and mammary arterial VSM, there was no change in BKCa {alpha}- or {beta}1-subunit mRNA after daily E2{beta} (Fig. 5; P > 0.4). Western blot analysis was also performed to determine whether there were changes in BKCa protein independent of mRNA. Neither {alpha}- or {beta}1-subunit protein was altered (P ≥ 0.3; data not shown).



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  		Fig. 5. Results of RT-PCR for BKCa {alpha}- and {beta}1-subunit mRNA in mesenteric arterial VSM from ovariectomized nonpregnant ewes after treatment with vehicle (n = 6) and daily E2{beta} (1 µg/kg; n = 5). A: results of RT-PCR for {alpha}- and {beta}1-subunits and MDH. B: results of densitometry in arbitrary units corrected for MDH mRNA. Values are means ± SE. Groups are not different for either {alpha}- or {beta}1-subunit. P > 0.4.

 
Myometrium. The myometrium from women and mice expresses BKCa (2, 7, 28), and E2{beta} appears to alter {alpha}- and/or {beta}1-subunit expression (3, 16, 51). Thus we also examined the effects of daily E2{beta} on myometrial samples that were available from animals in groups 1 and 2. Although daily E2{beta} did not affect either BKCa {alpha}-subunit mRNA (Fig. 6 ; P = 0.3) or protein (Fig. 7 ; P = 0.7), levels of {beta}1-subunit mRNA rose by 74% after daily E2{beta} (Fig. 6; P = 0.003), and this was paralleled by a proportionate rise in BKCa {beta}1-subunit protein (Fig. 7; P = 0.005).



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  		Fig. 6. Results of RT-PCR for BKCa {alpha}- and {beta}1-subunit mRNA in myometrium from ovariectomized nonpregnant ewes after treatment with vehicle (n = 6) and daily E2{beta} (1 µg/kg; n = 6). A: results of RT-PCR for {alpha}- and {beta}1-subunits and MDH. B: results of densitometry in arbitrary units corrected for MDH. Values are means ± SE. Groups are not different for {alpha}-subunit. P = 0.3; *P < 0.003 for increase in {beta}1-subunit after daily E2{beta}.

 


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  		Fig. 7. Immunoblot analysis of BKCa {alpha}- and {beta}1-subunit protein in myometrium from ovariectomized nonpregnant ewes after treatment with vehicle (n = 6) and daily E2{beta} (1 µg/kg; n = 6). A: immunoblot analysis for BKCa {alpha}- and {beta}1-subunit protein. B: analysis of densitometry in arbitrary units. Values are means ± SE. Groups are not different for {alpha}-subunit protein; P = 0.7. However, {beta}1-subunit protein increases significantly after daily E2{beta}; *P = 0.005.

 
Immunohistochemistry. RT-PCR and immunoblot analysis demonstrated the presence of both BKCa subunits in uterine arterial VSM and myometrium as well as increases in {beta}1-subunit expression in both tissues. This, however, does not provide information about their tissue distribution, nor does it provide insight into changes that might have occurred in the smaller resistance arteries within the myometrium. Therefore, we performed immunohistochemistry to examine the cellular distribution of BKCa {alpha}- and {beta}1-subunits in intact second generation uterine arteries, myometrium, and intramyometrial arteries from animals in groups 1 and 2.

BKCa {alpha}-subunit immunostaining was apparent throughout the media of the uterine artery but was not evident in the endothelium of either the vehicle-treated (Fig. 8C) or E2{beta}-treated (Fig. 8D) arteries. Consistent with the immunoblot analyses, there was no obvious effect of daily E2{beta} on {alpha}-subunit immunostaining in uterine arterial VSM. There also was diffuse immunostaining for the {beta}1-subunit in uterine arterial VSM but not in the endothelium (Fig. 8, E and F). In contrast to the {alpha}-subunit, {beta}1-subunit immunostaining increased dramatically throughout the media of arteries from ewes receiving daily E2{beta} (Fig. 8F).



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  		Fig. 8. Immunohistochemistry for BKCa {alpha}- and {beta}1-subunit protein in intact uterine arteries from nonpregnant ewes after treatment with vehicle (A, C, and E) and daily E2{beta} (1 µg/kg; B, D, and F) at x20 magnification. A and B: tissues incubated with nonimmune rabbit serum in absence of primary antisera and showing no immunostaining; m, media; L, vessel lumen; solid arrowhead, luminal endothelium. C and D: diffuse immunostaining for {alpha}-subunit protein throughout smooth muscle cells of media (m) that is unchanged after daily E2{beta}. There also is immunostaining in subendothelial cells (open arrowhead) that is unaffected by E2{beta}. Immunostaining for {alpha}-subunit was absent in luminal endothelium (solid arrowhead) of uterine arteries in presence and absence of E2{beta}. E and F: immunostaining was observed for {beta}1-subunit protein in smooth muscle cells in media (m); intensity was increased after daily E2{beta} (F). Immunostaining was also evident in subendothelial cells (open arrowhead). Adventitial tissue (a) did not show immunostaining. Immunostaining for {beta}-subunit was absent in luminal endothelium (solid arrowhead).

 
We performed immunostaining of the myometrium to examine not only the uterine smooth muscle cells but also the VSM of small intramyometrial branches of the uterine artery that represent resistance arteries. Immunostaining for the {alpha}-subunit was detected throughout the myometrium and media of the intramyometrial arteries (Fig. 9C) ; however, there was no apparent change in the intensity of immunostaining after daily E2{beta} (Fig. 9D). {beta}1-Subunit immunostaining was also evident throughout the myometrium and media of the intramyometrial arteries (Fig. 9E). In contrast to the {alpha}-subunit, immunostaining for {beta}1-subunit protein was markedly increased in the myometrium and VSM of intramyometrial arteries after daily E2{beta} (Fig. 9F). Thus the increase in {beta}1-subunit immunostaining in the media of intramyometrial arteries parallels that observed in the more proximal uterine arterial VSM (Fig. 8F).



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  		Fig. 9. Immunohistochemistry for BKCa {alpha}- and {beta}1-subunits in myometrium from nonpregnant ewes after treatment with vehicle (A, C, and E) and daily E2{beta} (1 µg/kg; B, D, and F) at x20 magnification. A and B: controls incubated with nonimmune rabbit serum without primary antibody and showing no immunostaining; solid arrowhead, intramyometrial arteries. C and D: diffuse immunostaining for {alpha}-subunit protein throughout myometrium and in smooth muscle of intramyometrial arteries (solid arrowhead) that was similar after vehicle (C) and daily E2{beta} (D) treatment. E and F: diffuse immunostaining for {beta}1-subunit protein in myometrium and media of intramyometrial arteries (solid arrowhead). In contrast to treatment with vehicle (E), there was marked increase in immunostaining within vascular and uterine smooth muscle after daily E2{beta} (F).

 

   DISCUSSION
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Acute and prolonged E2{beta} treatment increases blood flow to selected nonreproductive tissues, e.g., the heart and skin (23, 27, 44), and all reproductive tissues in nonpregnant, pregnant, and postpartum ewes (26, 27, 44). The uterine responses to E2{beta} have been studied in some detail and are associated with increases in the vascular synthesis of NO and cGMP (41, 43, 50, 54); however, the intracellular pathways involved are incompletely understood. In recent studies, Rosenfeld and colleagues (40, 42, 45, 46) reported that BKCa contribute to the terminal intracellular events responsible for E2{beta}-induced uterine vasodilation in nonpregnant and pregnant ewes and closely resemble that seen in the coronary circulation (12, 23, 58, 59). These data established a relationship between increases in BKCa activity and E2{beta}-mediated vasodilation in these vascular beds but did not address the issue of E2{beta}-mediated changes in VSM BKCa density or channel phenotype. In the present studies, E2{beta} did not acutely alter expression of either the BKCa {alpha}- or {beta}1-subunits in reproductive arterial VSM, and daily E2{beta} did not affect {alpha}-subunit expression in any tissue studied. However, daily E2{beta} selectively increased BKCa {beta}1-subunit mRNA and/or protein in arterial vascular and nonvascular smooth muscle from the reproductive tract but not in arterial VSM from a nonreproductive tissue. Thus neither prolonged nor acute E2{beta} appear to alter channel density in arterial VSM. However, there were E2{beta}-induced increases in arterial VSM {beta}1-subunit expression that could enhance BKCa sensitivity to estrogen exposure and changes in intracellular calcium, facilitate the uterine vascular responses to E2{beta}, decrease basal vascular tone, and, possibly, alter vascular sensitivity to vasoconstrictors (1, 5, 9, 11, 32).

BKCa are products of the slo gene and members of the voltage-gated K+ channel superfamily (53). They exhibit substantial phenotypic and functional diversity due to the expression of various isoforms of the constituent subunits (6, 15, 18, 29, 30, 52, 57). BKCa consist of four pore-forming {alpha}-subunits that may be closely associated with regulatory {beta}-subunits (18, 22). The {beta}1-subunit, one of several {beta}-subunit isoforms, is expressed exclusively in smooth muscle, including VSM as observed in the present study (34, 35). It regulates BKCa Ca2+ and voltage sensitivity, myogenic tone, and importantly, the binding of various pharmacological agents, including charybdotoxin and E2{beta} (13, 29, 55). Deletion of the {beta}1-subunit results in a loss of vascular responses to E2{beta} (5, 13, 55), implying an essential role in estrogen-mediated responses, an increase in basal vascular tone, and the development of hypertension (5, 32, 58). Furthermore, spontaneously hypertensive rats exhibit a decrease in VSM {beta}1-subunit expression (1). Thus variation in BKCa {alpha}- and {beta}1-subunit expression or stoichiometry in VSM, i.e., changes in number or phenotype, provides a modulator of vascular tone and reactivity (5, 32, 36, 52, 57). However, the mechanisms that regulate VSM BKCa subunit expression are unknown.

E2{beta} rapidly increases BKCa Po in VSM (18, 46, 55, 59); however, it is unclear whether E2{beta} also alters channel density in VSM. We observed the BKCa {alpha}-subunit in arterial VSM from ovine reproductive and nonreproductive tissues but not in the vascular endothelium, demonstrating cellular specificity within the vessel wall. E2{beta} exposure did not affect {alpha}-subunit mRNA and/or protein in VSM from either type of artery or in the media of small intramyometrial resistance arteries. Thus E2{beta} does not appear to change BKCa density in arterial VSM from a variety of tissues, and the increase in basal UBF and augmentation of acute UBF responses to E2{beta} observed after daily E2{beta} treatment (50) are unlikely due to changes in channel number. Alternatively, these responses to daily E2{beta} could reflect increases in uterine artery eNOS and nNOS expression and the capacity to synthesize more NO (41, 50, 54). Another consideration is that changes in the regulatory {beta}1-subunit are involved.

The {beta}1-subunit was also detected in VSM from all of the arteries examined but not the endothelium, providing additional evidence of cellular specificity for BKCa expression within the arterial wall and coexpression with the {alpha}-subunit. As with the BKCa {alpha}-subunit, acute E2{beta} did not alter {beta}1-subunit mRNA or protein. In contrast, daily E2{beta} increased {beta}1-subunit transcripts in VSM from both reproductive arteries that were studied and protein in uterine arterial VSM. Thus we report for the first time that prolonged E2{beta} treatment, as used in the present study, modifies the transcription and translation of the BKCa regulatory {beta}1-subunit in reproductive arterial VSM. The increase in uterine artery {beta}1-subunit in the absence of changes in {alpha}-subunit expression supports the thesis that channel stoichiometry, i.e., the {alpha}:{beta}1 ratio, in the absence of E2{beta} is less than 1:1 (52, 55) and increases after daily E2{beta}. However, we are presently unable to determine the actual changes in stoichiometry. Nonetheless, our findings suggest that this may be associated with increases in basal BKCa activity that contribute to the rise in basal UBF after daily E2{beta} (50) or enhance BKCa sensitivity to infused E2{beta} and augment the UBF responses to acute E2{beta} that occur after daily E2{beta} (13, 14, 41, 50). Although E2{beta} is a potent vasodilator in the coronary circulation and the BKCa are also involved, it is unclear whether acute coronary responses are similarly augmented after daily E2{beta} treatment (23). Notably, uterine vascular eNOS and nNOS also increase after a week of daily E2{beta} (41, 50, 54). Therefore, the augmented acute uterine responses to E2{beta} after daily E2{beta} may be due to the synergistic interaction between the simultaneous increases in NOS, VSM cGMP, and the {beta}1-subunit. Because the {beta}1-subunit also binds E2{beta} and appears necessary for E2{beta}-mediated responses (13, 14, 55), increases in the number of {beta}1-subunits may provide an alternative pathway to enhance the UBF responses. These questions deserve further study. Unlike the reproductive arteries, the mesenteric arterial VSM BKCa were unaffected by E2{beta}, resembling that recently seen with nNOS expression (41) and consistent with an absent blood flow response (27, 44). This could explain the tissue-specific responses noted earlier or reflect differences in the signaling cascade within reproductive and nonreproductive arteries because estrogen receptors are present in mesenteric arterial VSM (unpublished observations).

We also observed {beta}1-subunit protein in the media of small intramyometrial resistance arteries and documented a substantial increase in immunostaining after daily E2{beta}. Thus our observations in second and third generation uterine arteries (2–3 mm OD) can be extended to the smaller distal arterioles that actually modulate tissue vascular resistance and tissue blood flow, further supporting the importance of the VSM {beta}1-subunit in UBF responses to E2{beta} (13, 14). Interestingly, submillimolar concentrations of tetraethylammonium chloride infused into the uterine arterial circulation of pregnant ewes decrease basal UBF dose dependently (40, 42). Thus the increase in placental estrogen synthesis in pregnancy may serve to regulate BKCa expression in the uterine circulation, facilitating and maintaining the 40-fold rise in UBF that occurs in pregnancy and is essential for fetal growth and well being (49). It is unclear, however, whether this reflects increases in channel density, i.e., increases in the {alpha}-subunit, and/or changes in the {beta}1-subunit that modify basal channel activity and sensitivity to local estrogen. Studies are under way to address this.

Rosenfeld and colleagues (31, 39) previously reported that daily plus acute E2{beta} treatment attenuates systemic pressor responses to infused ANG II and {alpha}-agonists in nonpregnant animals. The mechanism for this remains unclear. Decreased pressor responses to both agonists also occur in normal pregnancy (8, 24, 38, 48). It was suggested that the increase in circulating estrogens in pregnancy might contribute to the attenuated vascular sensitivity to pressors (48, 49). The {beta}1-subunit knockout demonstrates increases in vascular tone and hypertension (32), and spontaneously hypertensive rats have decreases in VSM {beta}1-subunit expression (1, 9); thus E2{beta}-mediated changes in BKCa expression in the peripheral vasculature might account for the attenuated pressor responses after daily E2{beta} (17, 37). However, on the basis of the present results, this does not appear to be due to modification of the BKCa in the peripheral arterial vasculature because both the {alpha}- and {beta}1-subunits in mesenteric arterial VSM were unchanged by daily E2{beta}. Notably, mesenteric arterial VSM nNOS is also unaffected by daily E2{beta} (41). Because there is increasing evidence of tissue-specific responses to E2{beta}, this does not preclude changes in the BKCa phenotype or increases in Po in other peripheral arterial VSM, e.g., the skin (27, 44), which have not yet been examined.

We examined the myometrium to determine whether the E2{beta}-mediated changes in reproductive VSM also occurred in nonvascular smooth muscle from the reproductive tract. Neither BKCa {alpha}-subunit mRNA nor protein was affected by E2{beta}, providing additional evidence that E2{beta} does not alter channel density in nonpregnant animals. However, daily E2{beta} increased myometrial {beta}1-subunit transcripts and protein in a manner resembling the uterine arterial VSM. This is consistent with observations in mice when using a similar experimental paradigm (3); however, these investigators did not examine VSM or the {alpha}-subunit. They differ from the findings of Holdiman et al. (16), who observed increases in {alpha}-subunit in the mouse myometrium after E2{beta}. Importantly, the changes in the myometrium probably reflect the summation of responses in vascular and nonvascular smooth muscle. E2{beta} may also modulate differential splicing of the BKCa {alpha}-subunit, thereby modifying the channel isoform as well as the stoichiometric relationship between the {alpha}- and {beta}-subunits and thus channel function (16). This may represent another important regulatory mechanism in pregnancy whereby myometrial {beta}1-subunit expression and splice variants of the BKCa {alpha}-subunit (2, 3) increase and then decrease after parturition (28, 51). Thus BKCa activation may facilitate myometrial relaxation during pregnancy via estrogen-mediated effects because progesterone does not affect subunit expression (3).

We have provided the first evidence that daily E2{beta} exposure selectively regulates BKCa {beta}1-subunit expression in reproductive arterial VSM of nonpregnant sheep without altering the {alpha}-subunit and thus channel density. This suggests that changes in {beta}1-subunit expression and {alpha}:{beta}1 stoichiometry contribute to E2{beta}-mediated increases in basal and stimulated uterine and mammary blood flow by altering channel sensitivity and/or binding with E2{beta} without changing channel density (13, 55). This needs to be addressed in future studies. Notably, the increase in {beta}1-subunit expression parallels increases in vascular eNOS and nNOS (41, 50, 54), which could augment and optimize increases in UBF that are essential to embryo implantation in early pregnancy (49). If BKCa play a prominent role in regulating the uterine vasculature, and if changes in {beta}1-subunit expression modulate channel responsiveness and activity and thus basal and stimulated vascular resistance, it may be possible to design agents capable of regulating the relative number of {beta}1-subunits present and UBF in pregnancies with low UBF and/or hypertensive disorders (47).


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 ABSTRACT
 METHODS
 RESULTS
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 GRANTS
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These studies were supported by National Institutes of Health Grant HD-08783–29 and the George L. MacGregor Professorship in Pediatrics.


   ACKNOWLEDGMENTS
 
D. Nagar was a postdoctoral trainee in the Division of Neonatal-Perinatal Medicine at University of Texas Southwestern Medical Center at Dallas.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. R. Rosenfeld, Dept. of Pediatrics, Univ. of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9063 (e-mail: charles.rosenfeld{at}utsouthwestern.edu)

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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