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Cardiovascular Research 2002 53(3):650-661; doi:10.1016/S0008-6363(01)00428-X
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Endothelium-independent effect of estrogen on Ca2+-activated K+ channels in human coronary artery smooth muscle cells

Richard E Whitea,*, Guichun Hana, Melissa Maunzb, Christiana Dimitropouloua, Abdalla M El-Mowafyc, Robert S Barlowb, John D Catravasd, Connie Sneadd, Gerald O Carriera, Shu Zhua and Xiuping Yua

aDepartment of Pharmacology and Toxicology, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-2300, USA
bDepartment of Physiology and Biophysics, Wright State University School of Medicine, Dayton, OH 45435, USA
cDepartment of Applied Therapeutics, University of Kuwait, Safat 13110, Kuwait
dVascular Biology Center, Medical College of Georgia, Augusta, GA 30912, USA

rwhite{at}mail.mcg.edu

* Corresponding author. Tel.: +1-706-721-7582; fax: +1-706-721-2347

Received 15 May 2001; accepted 23 July 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Postmenopausal estrogen replacement therapy lowers the incidence of cardiovascular disease, suggesting that estrogens support cardiovascular function. Estrogens dilate coronary arteries; however, little is known about the molecular basis of how estrogen affects the human coronary circulation. The cellular/molecular effects of estrogen action on human coronary smooth muscle were investigated in the present study. Methods: Patch-clamp and fluorescent microscopy studies were performed on human coronary myocytes in the absence of endothelium. Results: Estrogen increased whole-cell currents over a range of membrane potentials, and further studies indicated that the large-conductance (186.5±3 pS), calcium- and voltage-activated potassium (BKCa) channel was the target of estrogen action. Channel activity was stimulated ~15-fold by nanomolar concentrations of 17β-estradiol, and this stimulation was reversed >90% by inhibiting cGMP-dependent protein kinase activity with 300 nM KT5823. 17β-Estradiol increased the level of cGMP and nitric oxide in human myocytes, and the stimulatory effect of estrogen on channel activity and NO production was reversed by inhibiting NO synthase with 10 µM NG-monomethyl-L-arginine. Conclusions: Our cellular and molecular studies identify the BKCa channel as a target of estrogen action in human coronary artery smooth muscle. This response to estrogen involves cGMP-dependent phosphorylation of the BKCa channel or a closely associated regulatory molecule, and further evidence suggests involvement of the NO/cGMP signaling system in coronary smooth muscle. These findings are the first to provide direct evidence for a molecular mechanism that can account for endothelium-independent effects of estrogen on human arteries, and may also help explain why estrogens reduce myocardial ischemia and stimulate coronary blood flow in patients with diseased coronary arteries.

KEYWORDS Coronary circulation; Gender; Hormones; Ion channels; K-channel; Smooth muscle


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Although cardiovascular disease has traditionally been considered a male problem, dysfunction of the heart and blood vessels accounts for nearly half of all female deaths annually. In fact, the number of women dying from cardiovascular disease, particularly coronary heart disease, has continued to increase [1]. During childbearing years women rarely suffer significant cardiovascular problems; however, early surgical menopause increases the risk of coronary heart disease, while estrogen therapy reduces this risk [2]. Meta-analyses of studies reveal that postmenopausal estrogen therapy is associated with a 35–50% reduced risk of coronary heart disease [1], yet the mechanism(s) for the cardiovascular protective effect of estrogen has (have) remained elusive. Because estrogen-induced alterations in serum lipids can account for only as much as one-third of the clinical benefits of female hormones, it is believed that direct vasodilatory effects of estrogen play an important role in reducing the incidence of cardiovascular disease [3].

Although vascular effects of female sex hormones were proposed over 100 years ago [4], knowledge of the cellular and/or molecular basis of estrogen-induced dilation of human blood vessels is far from complete. Clinical studies of postmenopausal women with coronary artery disease have revealed that acute (15–30 min) administration of estrogen ameliorates the symptoms of myocardial ischemia [5,6] and increases coronary blood flow [7]. Because the effects of estrogen on coronary flow were observed in women with dysfunctional coronary endothelium, it has been suggested that estrogen induces coronary artery relaxation via both endothelium-dependent mechanisms and direct (i.e., endothelium-independent) interaction with coronary smooth muscle. Moreover, estrogen, at concentrations within or near plasma levels, relaxes endothelium-denuded human coronary arteries [8,9]. Thus, it is becoming increasingly clear that estrogen can interact directly with human vascular smooth muscle to affect contractile tone. To date, however, no studies have investigated the effects of estrogen on single smooth muscle cells from human vessels.

The purpose of the present study was to characterize direct effects of estrogen on single myocytes from human coronary arteries. In order to preclude any potential influence from endothelial cells, human coronary smooth muscle cells were grown in culture and only contractile myocytes were employed in these studies. Both single-channel and perforated-patch whole-cell recordings revealed a specific target of 17β-estradiol in these human cells: the large-conductance, calcium- and voltage-activated potassium (BKCa) channel. Potassium efflux through this channel provides a powerful membrane repolarization to close voltage-dependent calcium channels and induce relaxation; therefore, we propose that the BKCa channel is a likely effector molecule mediating endothelium-independent effects of estrogen on human coronary arteries. In addition, this stimulatory effect of estrogen involves the NO/cGMP signaling system, which increases BKCa channel activity in smooth muscle and other cell types [10–16]. Therefore, the present results constitute a cellular mechanism that could account, at least in part, for estrogen-induced endothelium-independent relaxation of human coronary arteries observed in clinical studies.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Cell-culture
Human coronary artery smooth muscle cells were purchased from Clonetics/BioWhittaker, and were grown in smooth muscle growth medium (SmGM-2; BioWhittaker) supplemented with 5% fetal bovine serum. Cells (passages 3–7) were plated onto 12-mm cover slips for patch-clamp experiments. To insure that cells retained a contractile phenotype in culture, cover slips were treated with a high potassium depolarizing solution and cell contraction was observed under a microscope.

2.2. Patch-clamp studies
For cell-attached patch studies the recording chamber contained the following solution (mM): 140 KCl, 10 MgCl2, 0.1 CaCl2, 10 HEPES, and 30 glucose (pH 7.4; 22–25°C). Activity of single potassium channels was recorded (with pCLAMP 6.0.4, Axon Instruments) in cell-attached patches by filling the patch pipette (2–5 M{Omega}) with Ringer's solution (mM): 110 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2 and 10 HEPES. Voltage across the patch was controlled by clamping the cell at 0 mV with the high concentration extracellular potassium solution. In experiments recording potassium channel activity of inside-out patches the bathing solution exposed to the cytoplasmic surface of the membrane consisted of the following (mM): 60 K2SO4, 30 KCl, 2 MgCl2, 0.16 CaCl2, 1 BAPTA (pCa 7), 10 HEPES, 5 ATP, and 10 glucose (pH 7.4; 22–25°C). The pipette contained the high (symmetrical) 140 mM K+ solution listed above minus glucose. Currents were filtered at 1 kHz and digitized at 10 kHz. Average channel activity (expressed as number of channelsxsingle-channel open probability, NPo) in patches with multiple BKCa channels was determined as described previously [17]. NPo calculations were based on 10–15 s of continuous recording during periods of stable channel activity. Although channel activity was observed at a variety of membrane potentials, most single-channel data were recorded at a potential of +40 mV where BKCa channel openings are easily distinguished from other channel species to permit more accurate statistical analysis [18].

For perforated patch experiments the recording chamber contained a standard bath solution of the following composition (mM): 140 NaCl, 5 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, 30 glucose (pH 7.4; 22–25°C). To measure potassium currents the tip of the patch pipette (1–3 M{Omega}) was filled with a solution containing (mM): 60 K2SO4, 30 KCl, 5 MgCl2, 5 CaCl2, 5 HEPES and 40 MgSO4 (pH 7.4). The remainder of the pipette was back-filled with the same solution to which 6 mg/ml amphotericin B or nystatin (diluted by sonication from a 50-mg/ml stock in dimethylsulfoxide) was added. Voltage-clamp and voltage pulse generation were controlled with an Axopatch 200A patch-clamp amplifier (Axon Instruments), and data was acquired and analyzed with pCLAMP 6.0.4. Voltage-activated currents were filtered at 1 kHz and digitized at 10 kHz. Leakage currents were algorithmically subtracted using short duration, small amplitude negative prepulses.

2.3. Cyclic nucleotide measurements
Cyclic GMP was determined by enzyme immunoassay as previously described [19]. Cells, passages 3–7, were cultured in 48-well culture dishes at equal densities in an atmosphere of 5% CO2 at 37°C. Experiments were performed when cells reached 85–90% confluence. Cells were incubated in low-serum media (0.5–1%) for 18 h. Medium was removed and cells were washed three times with buffer (0.5 ml/well) containing 0.1% bovine serum albumin. Cells were then preincubated for 15 min at 37°C in buffer containing 0.1 mM 3-isobutyl-1-methylxanthine to inhibit phosphodiesterases. Estrogen (10–100 nM) was then added, whereas control cells were treated with an equivalent amount of vehicle (ethanol, total concentration always below 0.1%). Reactions were terminated after 15 min by removing the buffer and adding 0.5 ml of 0.1 N HCl for 30 min at room temperature. In preliminary studies, cell debris was solubilized in NaOH (1 mM, 0.4 ml) and protein content was measured by the method of Lowry et al. [20]. Consistently equal protein levels were found among cells. Accordingly, results were expressed as fmol cGMP/cell number.

2.4. Fluorescence studies
The cell-permeable form of the NO fluorescent indicator 4,5-diaminofluorescein diacetate (DAF-2 DA) was employed to examine production of NO within coronary myocytes [21,22]. Cells were placed on the stage of an Olympus IX70 inverted microscope and washed twice with Ringer's solution before loading with 1 µM DAF-2 DA (Ringer's solution; 45 min in the dark) diluted from a 5 mM stock solution in dimethylsulfoxide. After incubation, cells were washed several times with Ringer's solution. To measure fluorescence excitation light of 450–490 nm was supplied from a 50-W Hg lamp in order to observe specific fluorescence associated with the production of NO (500–530 nm). Images were captured with an Olympus C-3030 digital camera.

2.5. Drugs
BAPTA and KT5823 were purchased from Calbiochem. All other agents were purchased from Sigma.

2.6. Statistical analysis
All data were expressed as the mean±S.E. Statistical significance between two groups was evaluated by Student's t-test for paired data. Comparison between multiple groups was made by the one-way analysis of variance test. A probability of less than 0.05 was considered to indicate a significant difference.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Perforated-patch whole-cell recordings from single human coronary myocytes revealed non-inactivating outward currents that increased with membrane depolarization. Acute application of 100 nM 17β-estradiol (15–20 min) increased steady-state outward current at all positive voltages (Fig. 1A). A complete current–voltage relationship illustrating the stimulatory effect of 17β-estradiol is presented in Fig. 1B. Interestingly, the effect of 17β-estradiol was reversed by 10 nM iberiotoxin (5–10 min), a highly specific blocker of BKCa channels [23] (Fig. 1A,B). On average, iberiotoxin inhibited estrogen-stimulated current by 78.8±9% (n=4; +50 mV; P<0.03). A summary of the stimulatory effect of 100 nM 17β-estradiol on outward current is presented in Fig. 1C. These findings clearly demonstrate that estrogen stimulates outward current in human coronary smooth muscle cells in the absence of endothelium, and suggest involvement of BKCa channels.


Figure 1
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  		Fig. 1 Estrogen increases whole-cell currents in human coronary myocytes. (A) Perforated-patch recordings from the same myocyte before and 15 min after exposure to 100 nM 17β-estradiol, and then 10 min after cumulative addition of 10 nM iberiotoxin. Tracings were taken from a range of potentials (–50 to +50 mV), with the holding potential –60 mV. (B) The complete current (pA)–voltage (mV) relationship for steady-state outward current from the same cell as in (A). Treatment conditions are the same as described above for control, 17β-estradiol (17β-E), and 17β-estradiol+iberiotoxin (IBTx). (C) Average current (normalized as current density, pA/pF)–voltage relationship for coronary myocytes before and after 100 nM 17β-estradiol. Each point represents the mean of six cells±S.E.

 
Molecular studies on excised inside-out patches demonstrated that membrane electrical activity was dominated by a large-amplitude channel that conducted outward current. Studies in symmetrical gradients of potassium (140 mM) identified this protein as a potassium channel (Fig. 2A), and the average single-channel current–voltage relationship yielded a microscopic conductance of 186.5±3 pS (n=3–4 patches; Fig. 2B). Furthermore, this channel was stimulated by increasing [Ca2+] at the cytoplasmic face of the patch from 100 nM to 100 µM (Fig. 2C). Calcium-stimulated channel gating was inhibited by 1 mM TEA, which at this concentration is a specific inhibitor of BKCa channels (Fig. 2C). Given this specific biophysical and pharmacological profile, these experiments identify this protein as the BKCa channel. Subsequent studies on intact myocytes (cell-attached patches) revealed that estrogen produces a powerful stimulation of BKCa channel activity (Fig. 3). On average, 10 nM 17β-estradiol increased single BKCa channel activity (NPo) from 0.001±0.001 to 0.132±0.028 (+40 mV; n=5; P<0.04; Fig. 3A). Channel activity was also stimulated by 1 nM 17β-estradiol (NPo from 0.002±0.001 to 0.028±0.006; n=4; P=0.01), and 100 nM 17β-estradiol (NPo from 0.013±0.004 to 0.221+0.04; n=25; P<0.0001). The concentration dependency of the stimulatory effect of estrogen on BKCa channel activity is illustrated in Fig. 3B. In general, there was a 10–15-min latency before we observed estrogen-stimulated channel activity, and the effect of estrogen appeared to be maximal within 30–45 min. In nearly all patches this activity persisted until seal integrity was lost (generally after 90–120 min). Therefore, results from both whole-cell and single-channel studies demonstrate that estrogen opens BKCa channels in human coronary smooth muscle cells.


Figure 2
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  		Fig. 2 Human coronary myocytes express functional BKCa channels. (A) Tracings from the same inside-out patch recorded under symmetrical (140 mM) [K+] over a voltage range from –60 to +60 mV. Channel openings are either upward or downward deflections from baseline (closed) state (dashed line). (B) Average single-channel current (pA)–voltage (mV) relationship in symmetrical gradients of potassium. Each point represents the mean of three to four inside-out patches±S.E. (C) Representative traces from the same cell-attached patch (+40 mV; top panel; ~100 nM [Ca2+]) before and after excision into the inside-out patch configuration with [Ca2+]i=100 µM (middle panel). Tetraethyl-ammonium (TEA; 1 mM) reverses Ca2+-stimulated channel activity (bottom panel). Channel openings are upward deflections from the baseline (closed) state (dashed line).

 

Figure 3
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  		Fig. 3 Estrogen opens BKCa channels in human coronary smooth muscle cells. (A) Continuous 500 ms recordings from the same cell-attached patch (+40 mV) before (control) and 20 min after exposure to 10 nM 17β-estradiol. Channel openings are upward deflections from the baseline (closed) state (dashed line). (B) Summary of estrogen effect on BKCa channel activity. Bars represent average channel activity (NPo±S.E.) 15–25 min after 1, 10, or 100 nM 17β-estradiol. *Significant increase in channel activity above control (P<0.05).

 
Experiments on cell-attached patches revealed that the stimulatory effect of estrogen was mediated via the cGMP/PKG signal transduction pathway. As expected, 100 nM 17β-estradiol increased BKCa channel activity significantly (from 0.013±0.011 to 0.251±0.074; n=6); however, further addition of 300 nM KT5823, a membrane-permeable selective inhibitor of PKG [24], completely reversed the effect of estrogen within 10–20 min (NPo 0.017±0.008; n=6; P<0.002). The typical response to 17β-estradiol and KT5823 is illustrated by the traces in Fig. 4A, and the average effects are summarized in Fig. 4B. Furthermore, biochemical studies verified that estrogen increases cGMP accumulation in these cells in a concentration-dependent fashion. Cyclic GMP levels in human coronary myocytes were doubled by 10 nM 17β-estradiol, and nearly tripled by 100 nM 17β-estradiol (n=6–8; P<0.05; Fig. 5A). Functionally, cell-attached patch experiments demonstrated that increasing intracellular [cGMP] with a membrane-permeable derivative of cGMP, chlorophenylthio(CPT)-cGMP (100 µM; 20–30 min), mimicked the effect of 17β-estradiol on BKCa channels (Fig. 5B). On average, CPT-cGMP increased channel NPo by over 5-fold (from 0.027±0.018 to 0.174±0.061; +40 mV; n=5; P<0.05).


Figure 4
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  		Fig. 4 The stimulatory effect of estrogen on BKCa channels involves the cGMP-dependent protein kinase. (A) Representative recordings from the same cell-attached patch (+40 mV) before and 20 min after 100 nM 17β-estradiol, and 15 min after subsequent addition of 300 nM KT5823, an inhibitor of the cGMP-dependent protein kinase. Channel openings are upward deflections from the baseline (closed) state (dashed line). (B) Average effect of 100 nM 17β-estradiol (17β-E) and 300 nM KT5823 on cell-attached patches from human coronary smooth muscle cells. Each bar represents the average of six experiments±S.E. *Significant stimulation of channel activity above control levels (P<0.002). #Significant depression of estrogen-stimulated channel activity (P<0.002).

 

Figure 5
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  		Fig. 5 Cyclic GMP mediates the effect of estrogen on BKCa channels. (A) Estrogen increases cGMP accumulation in human coronary myocytes. Each bar represents the average levels of cGMP before and 15–20 min after 10 or 100 nM 17β-estradiol (n=6–8 determinations±S.E.). *Significant increase in cGMP levels compared to control (P<0.05). (B) Continuous 500 ms recordings from the same cell-attached patch (+40 mV) before (control) and 30 min after exposure to 100 µM CPT-cGMP. Channel openings are upward deflections from the baseline (closed) state (dashed line).

 
Estrogen increased cGMP accumulation in human coronary smooth muscle; however, the link between steroid and cyclic nucleotide is unknown. Further experiments suggested that nitric oxide might be involved in this process. FITC studies employing the membrane-permeable NO fluorescent indicator DAF-2 DA demonstrated that estrogen stimulates NO production in coronary myocytes (Fig. 6). These experiments revealed small basal amounts of DAF-2 fluorescence in unstimulated cells, but control studies indicated that these background levels were unchanged after over 2 h of DAF-2 treatment (data not shown). In contrast, acute exposure to 100 nM 17β-estradiol (15 and 30 min) clearly increased NO-induced fluorescence in coronary myocytes (Fig. 6A). To exclude potential nonspecific effects of DAF-2 or estrogen on fluorescence intensity, myocytes were exposed to 10 µM NG-monomethyl-L-arginine (L-NMMA; 20 min), an inhibitor of NO synthase. In the presence of L-NMMA, 100 nM 17β-estradiol did not increase NO fluorescence (Fig. 6B). These fluorescence studies were completely consistent with further patch-clamp experiments demonstrating that 10 µM L-NMMA (10 min) completely reversed the stimulatory effect of 17β-estradiol on BKCa channel activity (Fig. 7A). In these experiments 100 nM 17β-estradiol increased channel NPo from 0.017±0.012 to 0.247±0.077, but L-NMMA reduced channel NPo back to control levels (0.013±0.009; n=8; P<0.002). Involvement of the NO/cGMP/PKG cascade is further indicated by the combination of agents employed on the same myocyte, as illustrated in Fig. 7B. In this experiment the stimulatory effect of 100 nM 17β-estradiol (NPo from 0.096 to 0.561) was inhibited by 10 µM L-NMMA (NPo 0.000). This inhibition was overcome by 100 µM CPT-cGMP (NPo increased to 0.394), but (like estrogen) activity was again abolished by inhibiting PKG activity with 300 nM KT5823 (NPo 0.000). On average, KT5823 completely reversed cGMP-stimulated channel activity (NPo from 0.174±0.061 to 0.001±0.001; n=4; P<0.03).


Figure 6
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  		Fig. 6 Estrogen stimulates NO production in human coronary myocytes. Images are taken from FITC studies employing the NO fluorescence indicator DAF-2 DA (1 µM). Myocytes were incubated with DAF-2 DA for 45 min, and then basal levels of fluorescence were monitored for an additional 20 min prior to addition of estrogen. (A) Images of the same coronary myocytes before and then 15 or 30 min after exposure to 100 nM 17β-estradiol (17β-E). (B) Images from another population of coronary myocytes that was treated with 10 µM L-NMMA for 20 min before cumulative addition of 100 nM 17β-estradiol (15 or 30 min).

 

Figure 7
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  		Fig. 7 Inhibiting NO production reverses the effect of estrogen on BKCa channel activity. (A) Each bar represents the average channel open probability obtained from cell-attached patches (+40 mV) before and 15–20 min after 100 nM 17β-estradiol, and 10 min after subsequent addition of 10 µM L-NMMA. *Significant increase in channel activity compared to control (n=8; P<0.002). #Significant inhibition of estrogen-stimulated channel activity (n=8; P<0.002). (B) Activity plot of BKCa channel open probability in a single cell-attached patch before and 20 min after 100 nM 17β-estradiol, and then 10 min after addition of 10 µM L-NMMA. Inhibition was overcome by 100 µM CPT-cGMP (30 min), but subsequent exposure to 300 nM KT5823 (15 min) abolished cGMP-stimulated activity. Vertical bars are channel activity during a 100-ms test pulse to +40 mV. Recording time under each condition was 7–8 s, as indicated on the time axis. Breaks in the time axis represent drug incubation periods when channel activity was not recorded. Period of drug exposure is indicated by horizontal lines above the histogram.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Increasing clinical evidence indicates that estrogen dilates coronary arteries. For example, in female patients estrogen improves endothelium-dependent epicardial coronary vasomotor responses [25], and also increases blood flow by dilating coronary arteries with damaged endothelium [7]. Interestingly, acute (15 min) intravenous administration of conjugated estrogens also enhances coronary blood flow responses in males with coronary heart disease [26], thus underscoring the potentially broad therapeutic application of estrogen derivatives to treat cardiovascular disorders. The fact that estrogen dilates coronary arteries with damaged endothelium suggests a direct, endothelium-independent effect on coronary smooth muscle cells, and this suggestion is supported by in vitro studies demonstrating that estrogen relaxes human coronary arteries after endothelium denudation [8,9]. In the present study we observed effects of estrogen on single myocytes from human coronary arteries, thus providing direct cellular/molecular evidence that estrogen produces endothelium-independent responses in human coronary smooth muscle. These findings from human cells are supported by previous studies demonstrating that estrogen induces endothelium-independent relaxation of coronary arteries from rabbits [27] or pigs [17]. However, the effects of estrogen on blood vessels are species-dependent, and are believed to require an intact endothelium in rat coronary [15] or cerebral [28] arteries.

The present findings from both whole-cell (perforated-patch) and single-channel recordings demonstrate that the BKCa channel is a target of estrogen action in human coronary myocytes. Whole-cell currents were increased by 17β-estradiol, and this effect was inhibited by iberiotoxin, a highly selective blocker of BKCa channels. Furthermore, single-channel patch studies clearly identified BKCa channels in these human myocytes, and demonstrated that estrogen increases the open probability of these channels dramatically. These studies are consistent with previous reports indicating that the BKCa channel is the predominant potassium channel in primary myocytes from human coronary arteries [29]. Because of its large conductance and high density of expression, this channel plays an important role in setting and maintaining resting membrane potential of coronary smooth muscle [30], and also mediates coronary relaxation induced by a variety of vasoactive substances [14,31]. We had demonstrated previously that blockade of BKCa channels with iberiotoxin inhibits estrogen-induced relaxation of porcine coronary arteries by ~90% [17]. Furthermore, estrogen-induced reduction of ischemia and infarct size in canine hearts is attenuated by iberiotoxin [32], as is estrogen's ability to lower intracellular [Ca2+] in porcine coronary arteries [33]. In addition, we recently reported that ~70% of estrogen-mediated ovine uterine artery vasodilation is mediated by BKCa channel activity [34]. Thus, evidence from both in vivo and in vitro studies supports the contention that BKCa channels mediate a significant, if not majority, portion of estrogen-induced vascular relaxation in the coronary and other circulations. More directly, estrogen opens BKCa channels in single myocytes from porcine coronary [17,35] or ovine uterine [34] arteries in the absence of endothelium. Therefore, it is clear that BKCa channels are important targets of estrogen action in vascular smooth muscle from humans and other species.

Contraction of vascular smooth muscle is triggered by an increase in intracellular [Ca2+], and reduction of this calcium is, therefore, an important means of inducing vasodilation. Estrogen decreases [Ca2+]i in porcine coronary arteries [33] and inhibits contraction of rabbit coronary arteries [27] by decreasing influx of extracellular calcium across the plasma membrane, and these responses do not appear to involve specific effects on intracellular calcium release mechanisms. Previous studies had also indicated that estrogen inhibits calcium influx in porcine coronary arteries without changing the sensitivity of the contractile elements for calcium [36], and also depresses calcium currents in a vascular smooth muscle cell line [37]. In addition, 17β-estradiol antagonizes the effect of calcium channel agonists on coronary perfusion pressure [38]. Collectively, these studies suggest that estrogen acts as a physiological antagonist of calcium channel activity to suppress Ca2+ influx into coronary artery smooth muscle cells and promote vasodilation. Opening of BKCa channels is a very powerful, albeit indirect, means of reducing calcium influx via voltage-sensitive calcium channels, and the fact that iberiotoxin attenuates the ability of estrogen to suppress calcium influx in coronary arteries [33] underscores the importance of BKCa channels in mediating a significant portion of this vasodilatory response mechanism. Our findings in human coronary myocytes strongly suggest that that ability of estrogen to reduce myocardial ischemia and increase coronary blood flow in postmenopausal women and in men is related to its stimulatory effect on these potassium channels (as it is in canine hearts [32]); however, other cellular mechanisms (e.g., direct effect on calcium channels; release of endothelial vasoactive factors in healthy coronary arteries) are probably involved as well.

The transduction processes linking estrogen to ion channel activity are not well characterized, but evidence from the present study has identified some important elements in this signaling cascade. We found multiple evidences that estrogen stimulates the cGMP second messenger system in human coronary smooth muscle cells: (1) 17β-estradiol increases cGMP accumulation; (2) cyclic GMP, like estrogen, stimulates BKCa channel activity; and (3) an inhibitor of PKG activity reverses the stimulatory effect of either 17β-estradiol or cGMP on BKCa channel activity. It is reported that acute (30 min) exposure to 17β-estradiol increases cGMP accumulation in human coronary arteries, but cGMP was measured only in arteries with an intact endothelium [8]. In contrast, we measured estrogen-stimulated cGMP production in human myocytes and found that estrogen increases cGMP levels in the absence of endothelium. These findings are consistent with previous studies demonstrating that 17β-estradiol stimulates cGMP production in endothelium-denuded porcine coronary arteries [35]. Activation of the cGMP/PKG signaling cascade stimulates BKCa channel activity in non-human coronary artery smooth muscle [14–16], but the present study provides the first direct evidence that cGMP-stimulated phosphorylation opens BKCa channels in human coronary myocytes. Furthermore, our findings with KT5823, which at the concentration employed inhibits PKG selectively [24], imply that cGMP-dependent phosphorylation mediates the stimulatory effect of 17β-estradiol on K+ channel activity, as it does in porcine coronary arteries [17,35] or pancreatic β-cells [39].

At present, it is unclear how estrogen, in the absence of endothelium, stimulates cGMP production in coronary smooth muscle; however, our findings from both fluorescence and patch-clamp studies indicate that 17β-estradiol stimulates NO production in human coronary artery smooth muscle cells. Estrogen-stimulated NO production was detected directly with DAF-2, a fluorescent NO indicator. Moreover, the effect of estrogen on NO production and on BKCa channel activity was inhibited and/or reversed by blocking NO synthase activity with L-NMMA. Taken together, these findings strongly suggest that estrogen increases NO production in human coronary artery smooth muscle cells by stimulating NOS activity. The identity of the NOS isoform involved in this process remains unknown, but it is clearly an endothelium-independent mechanism as estrogen-induced NO production was observed in cultured human myocytes. Support for this mechanism of action is gained from previous studies of endothelium-denuded human [8] or porcine [17,35] coronary smooth muscle in which 17β-estradiol induced relaxation associated with cGMP and/or NO. Other studies also indicate that vascular effects of estrogen involve NO [15,32,34,40,41]. Alternatively, micromolar concentrations of 17β-estradiol open BKCa channels in artificial expression systems in the absence of endothelium, NO, or cGMP [42]; however, this mechanism cannot account for the observed action of estrogen in human coronary myocytes because the effect of 17β-estradiol was inhibited by blocking PKG or NOS activity. If estrogen interacted with BKCa channel proteins directly, then these inhibitors would have been without effect. Furthermore, numerous other studies indicate that both endothelium-dependent and endothelium-independent vascular effects of estrogen are mediated primarily by generation of intermediary intracellular signals [3,8,15,17,28,32,34,35,40,41]. Therefore, if direct estrogen–BKCa channel interaction occurs under physiological conditions, this interaction must play a comparatively minor role in estrogen-induced vasodilation.

In summary, the present findings provide evidence from both cellular and molecular studies that estrogen opens BKCa channels in human coronary artery smooth muscle. Because this stimulation occurred within minutes, not hours, it is most likely a non-genomic effect of estrogen, which would be consistent with previous clinical studies demonstrating acute effects of estrogen on coronary blood flow and/or relief of myocardial ischemia in patients of both sexes [5–7,26]. Involvement of the cGMP/PKG signaling cascade in the response to 17β-estradiol is well-supported by both biochemical and functional studies, as well as by previous studies on human [8] and non-human [17,35] coronary smooth muscle. A potential role for NO in the response of human coronary smooth muscle to estrogen is also indicated; however, the mechanism whereby estrogen stimulates endothelium-independent NO production remains to be clarified. Nonetheless, these findings constitute a cellular mechanism that can help explain the molecular basis of how estrogen induces endothelium-independent dilation of human coronary arteries. This phenomenon may be particularly important in helping to maintain coronary blood flow in hearts with diseased or dysfunctional coronary arteries, and would thereby contribute to the reduction of cardiovascular morbidity and mortality associated with postmenopausal estrogen replacement therapy [1].

Time for primary review 22 days.


   Acknowledgements
 
This work was supported by grants from the American Heart Association (R.E.W.; G.O.C.), the American Heart Association (Ohio-West Virginia Affiliate; M.M. and R.E.W.) and the National Heart, Lung, and Blood Institute (HL54844, R.E.W.; HL64779, R.E.W. and G.O.C.; HL52958, J.D.C.) We thank Ms. Alexa Dilberger (Clonetics Corp.) for her assistance in obtaining human coronary artery smooth muscle cells.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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