This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by White, R. E. Articles by Falvo Lang, J. L. Search for Related Content PubMed PubMed Citation Articles by White, R. E. Articles by Falvo Lang, J. L.

(Circulation Research. 1995;77:936.)
© 1995 American Heart Association, Inc.

Articles

Estrogen Relaxes Coronary Arteries by Opening BK_Ca Channels Through a cGMP-Dependent Mechanism

Richard E. White, David J. Darkow, Jessica L. Falvo Lang

From the Department of Physiology & Biophysics, Wright State University School of Medicine, Dayton, Ohio.

Correspondence to Richard E. White, PhD, Department of Physiology and Biophysics, Room 158 Biological Science Bldg, Wright State University School of Medicine, Dayton, OH 45435. E-mail rwhite@sirius.bio.wright.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Women rarely suffer cardiovascular dysfunction before menopause, but by the age of 65 a woman becomes as vulnerable to cardiovascular mortality as a man. It has been proposed that estrogens protect against cardiovascular disease; however, the physiological basis of estrogen protection is unknown. In the present study the mechanism of estrogen-induced relaxation of coronary arteries was investigated at the tissue, cellular, and molecular levels. Tissue studies demonstrated that 17ß-estradiol relaxes porcine coronary arteries by an endothelium-independent mechanism involving K⁺ efflux, and subsequent studies employing the patch-clamp technique confirmed that estrogen stimulates K⁺ channel gating in coronary smooth muscle. Perforated-patch recordings from metabolically intact coronary myocytes revealed that 17ß-estradiol more than doubles steady state outward currents in these cells at positive voltages. Studies of on-cell patches demonstrated a potent stimulatory effect of 17ß-estradiol on the gating of the large-conductance, Ca²⁺- and voltage-activated K⁺ (BK_Ca) channels, while 17-estradiol had no effect. Furthermore, blocking BK_Ca channels in intact arteries inhibited estrogen-induced relaxation. The effect of 17ß-estradiol on BK_Ca channels was blocked by inhibiting cGMP-dependent protein kinase (PKG) activity and was mimicked by exogenous cGMP or by stimulating PKG activity. Therefore, we propose that 17ß-estradiol relaxes coronary arteries by opening BK_Ca channels via cGMP-dependent phosphorylation. This novel mechanism could account for the hypotensive effect of estrogens and help explain, at least in part, why postmenopausal estrogen therapy lowers the risk of cardiovascular disease.

Key Words: estradiol • coronary artery • BK_Ca channel • cGMP • Ca²⁺-activated K⁺ channel

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cardiovascular disease is often considered a predominantly male health problem because only 10% of women suffer significant cardiovascular dysfunction during childbearing years. Although premenopausal women have a 50% lower risk of developing coronary artery disease, a 66% lower risk of stroke, and a 33% lower risk of sudden cardiac death than do males of comparable age, a woman’s chance of suffering cardiovascular disease rises to that of males within a few years after the onset of menopause.^{1 2} Thus, coronary heart disease is the most common and most lethal cardiovascular event for both sexes. Because there is no pathophysiological basis for this increasing risk after menopause, it has been proposed that estrogens delay and/or prevent progressive deterioration of cardiovascular function to keep premenopausal women "hemodynamically younger" than men of the same age.² Consonant with this hypothesis are studies demonstrating that postmenopausal estrogen replacement therapy reduces a woman’s risk of suffering myocardial infarction by 50% to 70%³ ; however, the mechanism(s) underlying estrogen’s protective effect is unknown. In most cases estrogen therapy lowers low-density lipoprotein cholesterol and increases high-density lipoprotein cholesterol levels, but the opposite effects may also occur.⁴ In fact, much of the total cardiovascular benefit of estrogen appears to be independent of changes in plasma lipoproteins.^{3 5}

Ovarian steroids are vasoactive substances, and there is now increasing interest in the hemodynamic effects of estrogens. For example, systemic vascular resistance and diastolic pressure both decrease during pregnancy when estrogen levels are on the rise.⁶ Interestingly, male transsexuals experience a 20% decline in peripheral resistance and a significant decrease in diastolic pressure after estrogen treatment.⁷ Furthermore, many manifestations of menopause, eg, cessation of menses and "hot flushes," can be attributed to changes in blood flow.⁸ Thus, there is substantial evidence that estrogens modulate circulatory hemodynamics; however, there is a paucity of data concerning the cellular or molecular basis of estrogen action on vascular smooth muscle. For example, 17ß-estradiol enhances cardiac blood flow by promoting coronary vasodilation,⁹ but the mechanism of action remains undefined. One clue was provided in 1979 when it was suggested that estrogen might stimulate K⁺ conductance in coronary arteries,¹⁰ and a more recent study has demonstrated that estrogen increases cyclic nucleotide levels in these vessels.¹¹ Now fifteen years later, the present study reconciles these results into a unifying mechanism for estrogen-induced coronary vasodilation that couples cGMP-dependent phosphorylation to K⁺ channel activity. These findings provide a plausible molecular mechanism that could underlie estrogen’s ability to stimulate organ blood flow and lower systemic vascular resistance, thereby contributing to the overall cardiovascular benefit of estrogen therapy.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Arterial Tension Studies
Fresh porcine hearts from castrated males or gilts were obtained from local abattoirs. The left anterior descending (LAD) coronary artery was excised and placed into ice-cold Krebs-Henseleit buffer solution of the following composition (in mmol/L): NaCl 122, KCl 4.7, NaHCO₃ 15.5, KH₂PO₄ 1.2, MgCl₂ 1.2, CaCl₂ 1.8, glucose 11.5, pH 7.2. Arteries were kept on ice during transport to the laboratory. Two 4- to 5-mm rings (2 to 4 mm in diameter) were obtained from each LAD coronary artery and prepared for isometric contractile-force recordings as described previously.¹² To control for possible indirect effects of endothelium-derived vasoactive factors, the endothelium was removed by rubbing the intimal surface. Rings were mounted on two triangular tissue supports, with one support fixed to a stationary glass rod and the other attached to a force-displacement transducer connected to a physiograph (Gould Instrument Systems, Inc). The tissue-bathing solution was the modified Krebs-Henseleit buffer (37°C) described above, oxygenated continuously with 97% O₂/3% CO₂. Coronary ring preparations were equilibrated for 90 minutes under an optimal resting tension of 2.0 g, and fresh solution was added every 30 minutes. Preparations were exposed to maximally effective concentrations of a contractile agonist to insure stabilization of the muscle. After agonist removal and reequilibration, a contractile agonist was reapplied to the tissue bath. After the response reached a stable, maximum level, 17ß-estradiol was added to the bathing medium. All drug solutions were prepared fresh daily. For high [K⁺] solutions NaCl was reduced to maintain normal osmolarity.

Cell Isolation
Myocytes were isolated by a modification of a procedure described previously.¹³ The endothelium was removed by rubbing the intimal surface of the artery, and the adventitia was carefully dissected away. Approximately 2 cm of media was then cut into 1-mm strips and placed in test tubes containing a low calcium dissociation medium of the following composition (in mmol/L): NaCl 110, KCl 5, CaCl₂ 0.16, MgCl₂ 2, HEPES 10, NaHCO₃ 10, KH₂PO₄ 0.5, NaH₂PO₄ 0.5, glucose 10, EDTA 0.49, and taurine 10, pH 6.9. Phenol red was omitted because it is a weak estrogen receptor agonist. Media strips were incubated at 37°C in 5 mL of the above solution with 6.33 mg papain, 4 mmol/L dithiothreitol, and 0.2% BSA. After 30 minutes of gentle shaking, strips were titurated and the enzyme activity was diluted by adding excess enzyme-free solution. The solution was then removed and centrifuged at low speed for 15 minutes. The resultant pellet was then resuspended in fresh medium and kept at 4°C. Experiments were performed within 6 to 8 hours after cell dissociation.

Patch-Clamp Studies
Several drops of cell suspension were placed in a recording chamber (Warner Instruments Corp) containing a solution of the following composition (in mmol/L): NaCl 140, KCl 5, MgCl₂ 2, CaCl₂, 2, HEPES 20, glucose 20 (pH 7.2; 22°C to 25°C). To measure K⁺ currents the tip of a patch pipette (1 to 3 M) was filled with a solution containing (in mmol/L): KCH₃SO₃ 90, KCl 40, MgCl₂ 5, and HEPES 20 (pH 7.4). The remainder of the pipette was backfilled with a similar solution to which 200 µg/mL nystatin (diluted by sonication from a 50-mg/mL stock in DMSO) was added. This nystatin–perforated-patch technique provides measurement of stable whole-cell currents without disrupting cytoplasmic concentrations of divalent cations or metabolites.¹⁴ Cells were studied only if the voltage drop across the series resistance could be reduced to 5 mV within 10 to 20 minutes after forming a gigaohm seal. Voltage-clamp and voltage-pulse generation were controlled with an Axopatch 200A patch-clamp amplifier (Axon Instruments, Inc), and data were acquired and analyzed with pCLAMP 6.0.1 (Axon Instruments, Inc). Voltage-activated currents were filtered at 2 kHz and digitized at 10 kHz. Leakage currents were subtracted digitally. Single K⁺ channels were measured in cell-attached patches by filling the patch pipette (2 to 5 M) with the standard bath solution minus glucose and making a gigaohm seal on a single myocyte. Voltage across the patch was controlled by clamping the cell at 0 mV with a high-concentration K⁺ extracellular solution containing (in mmol/L): KCl 140, MgCl₂ 10, CaCl₂ 0.1, HEPES 10, and glucose 30 (pH 7.2). Currents were filtered at 2 kHz and digitized at 10 kHz. Average channel activity (NPo) in patches with multiple large-conductance, Ca²⁺- and voltage-activated K⁺ channels (BK_Ca channels) was determined by:

where Po is the single-channel open-state probability, T is the duration of the recording, tj is the duration of j=1,2, ... n channel openings, J is the number of channels open for duration tj, and N is the maximal number of simultaneous channel openings observed when Po was high.

Drugs
Papain, dithiothreitol, nystatin, prostaglandin (PG)F₂, 17-estradiol, and 8-bromo-cGMP were purchased from Sigma Chemical Co. 17ß-estradiol, KT5823, and BAPTA-AM were purchased from Calbiochem. Charybdotoxin was purchased from Peninsula Laboratories. Iberiotoxin was purchased from Research Biochemicals International. Okadaic acid was purchased from LC Laboratories. Rp-8-CPT-cGMPS and Sp-8-CPT-cGMPS were purchased from Biolog Life Science. Stock solutions of estradiol, PGF₂, and KT5823 were made with 50% DMSO, and okadaic acid was dissolved in ethanol. These stock solutions were diluted into the appropriate bath solution with the total concentration of solvent never exceeding 0.1%. All other drugs were prepared with distilled water.

Statistical Analysis
Data from tissue studies were expressed as the percentage of maximum relaxation, and all other data were expressed as the mean±SEM. 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 ANOVA, with a post hoc Bonferroni test to determine significant differences among the data groups. A value of P<.05 was considered to indicate a significant difference.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The effects of estrogen on coronary artery function were explored at the tissue, cellular, and molecular levels. Isometric contractile-force recordings from ring preparations of intact arteries revealed a potent relaxing effect of 17ß-estradiol in the absence of a functional endothelium; however, the magnitude of this relaxation depended on the contractile agent employed (Fig 1). In normal (5 mmol/L) extracellular [K⁺], 5 µmol/L 17ß-estradiol relaxed arteries precontracted with either 1 µmol/L PGF₂ (100% relaxation, n=5) or 10 µmol/L histamine (89.3±4% relaxation, n=4). Although nanomolar concentrations of 17ß-estradiol produce significant relaxation of coronary arteries in vitro, maximum relaxation is not achieved until the concentration approaches the micromolar range.¹¹ To insure consistent, maximal responses subsequent experiments employed 5 to 10 µmol/L 17ß-estradiol. Interestingly, arterial contractions induced by depolarization with high extracellular [K⁺] were less sensitive to estrogen than contractions in normal [K⁺] (Fig 1A). 17ß-Estradiol relaxed contractions due to 30 mmol/L KCl by 52.4±7% (n=5). Moreover, relaxation was only 22.8±3% (n=9) in arteries precontracted with 80 mmol/L extracellular [K⁺] (Fig 1B). These findings indicate that estrogen acts on coronary smooth muscle directly to produce relaxation; however, this response is reduced significantly (P<.0001) as extracellular [K⁺] is raised beyond physiological levels. These findings suggested that estrogen-induced coronary relaxation requires K⁺ gradients suitable for K⁺ efflux, stimulation of which would lead to repolarization and subsequent relaxation.

View larger version (21K): [in this window] [in a new window]   Figure 1. Relaxation of porcine coronary arteries by 17ß-estradiol requires physiological gradients of K+. A, Isometric contractile-force recordings from endothelium-denuded arteries precontracted with 30 mmol/L or 80 mmol/L KCl (at arrows). Relaxation to 5 µmol/L 17ß-estradiol (17ß-E, at arrow) required 30 minutes to reach maximum levels. The broken line indicates baseline tension. B, Comparison of estrogen-induced coronary relaxation after precontraction with 10 µmol/L histamine and 30 or 80 mmol/L KCl. *Significant (P<.0001) difference compared with relaxation of arteries precontracted with histamine.

Direct evidence that outward currents are stimulated by estrogen is presented in Fig 2. Addition of 5 µmol/L 17ß-estradiol more than doubled steady state outward current at all positive voltages (170±39% at +40 mV; n=4, P=.02) without shifting the threshold of activation (Fig 2B), while 10 µmol/L 17-estradiol had no effect (Fig 2A; n=5). As seen in Fig 2A, these currents activated slowly and did not inactivate during the 200-millisecond depolarization. In addition, 5 nmol/L 17ß-estradiol stimulated outward currents (+40 mV) by 6.2±1.4% (n=2), and currents were increased 55.5±20% (n=3) by 20 nmol/L 17ß-estradiol. Charybdotoxin reversed the effect of 20 nmol/L 17ß-estradiol completely (Fig 2C) and also inhibited nonstimulated currents significantly. On average, 50 nmol/L charybdotoxin decreased steady state outward currents by 61±8% (+40 mV, n=5, P=.02), whereas glibenclamide, an ATP-sensitive K⁺ channel blocker (10 µmol/L, n=5), or 4-aminopyridine, a delayed rectifier K⁺ channel blocker (4 mmol/L, n=3), had no significant effect. Like other vascular smooth muscle cells, coronary myocytes express several K⁺ channels that conduct outward current; however, given these kinetics and sensitivity to charybdotoxin, it seemed likely that BK_Ca channel activity comprised the major portion of outward currents. A definitive identification of channel species was made by measuring single channel currents. Recordings from membrane patches revealed at least three distinct ion channels that carried outward current; however, channel activity was dominated by a prominent, high-conductance (119±14 pS; n=5 to 11; Fig 2D) channel that was blocked by 1 mmol/L tetraethylammonium, insensitive to 10 µmol/L glibenclamide, and stimulated by increasing Ca²⁺ at the cytoplasmic face of the membrane. This biophysical and pharmacological profile is consistent with that of a BK_Ca channel. Coronary myocytes express a high density of these channels¹⁵ and also possess high-affinity binding sites for [³H]estradiol.¹⁰ Therefore, it was possible that these molecules were involved in estrogen relaxation of coronary arteries.

View larger version (21K): [in this window] [in a new window]   Figure 2. 17ß-Estradiol–stimulated steady state outward currents in metabolically intact coronary myocytes. A, Steady state outward currents (+40 mV; holding potential -60 mV) after 1-hour exposure to 10 µmol/L 17-estradiol (17-E) or a 30-minute exposure to 5 µmol/L 17ß-estradiol (17ß-E). The broken line indicates baseline current. B, Complete current (pA)-voltage (mV) relation for steady state outward current of another cell before and 30 minutes after addition of 5 µmol/L 17ß-estradiol. C, Complete current-voltage relation for steady state outward current before and after a 1-hour exposure to 20 nmol/L 17ß-estradiol. The effect of 17ß-estradiol was reversed completely by 30 nmol/L charybdotoxin (CTX). D, Biophysical profile of single BKCa channel conductance, as determined from the single-channel current-voltage relationship in cell-attached patches.

This hypothesis was verified by experiments demonstrating that estrogen stimulates the gating of single BK_Ca channels (Fig 3). Under control conditions (cell-attached patch, membrane voltage, 0 to +40 mV), channel openings were infrequent. In contrast, 5 to 10 µmol/L 17ß-estradiol increased BK_Ca channel activity markedly in 21 of 25 cell-attached patches (membrane voltage, +40 mV). Of the 21 patches that responded to estrogen, only 1 contained a single BK_Ca channel (Fig 3A). In this patch 17ß-estradiol increased the probability of the channel’s being open by two orders of magnitude (Po_control=.003; Po_estrogen=.336). Furthermore, in the 20 patches with multiple BK_Ca channels (eg, Fig 3B) 17ß-estradiol also stimulated channel activity, as estimated by NPo, by about two orders of magnitude (.0057±.01 to .5089±.15; P=.002). This effect often required 30 to 60 minutes to reach a maximum level, as does estrogen-induced relaxation of arteries in vitro¹¹ and in vivo.¹⁶ Alternatively, it was possible that increased channel activity after long incubation periods might actually be due to accumulation of myoplasmic Ca²⁺. To rule out this possibility, cells were preloaded with 10 to 50 µmol/L BAPTA-AM to buffer [Ca²⁺]_i. Treating cells with BAPTA-AM for 20 minutes did not prevent estrogen’s stimulation of BK_Ca channel activity (n=5 cell-attached patches). These findings from single myocytes suggested that estrogen-induced relaxation of coronary arteries was due to enhanced gating of BK_Ca channels. If this hypothesis were correct, then blockade of BK_Ca channels in intact arteries should inhibit estrogen-induced relaxation. Treating coronary arteries with 10 nmol/L iberiotoxin, a highly selective inhibitor of BK_Ca channels,¹⁷ induced a contraction that, as predicted, was relatively resistant to estrogen (Fig 3C). Thirty-minute exposure to 17ß-estradiol in 5 mmol/L extracellular [K⁺] produced only minimal (12.4±2%; n=6) relaxation of iberiotoxin-induced contraction, a response similar to the blunted effect observed when arteries were precontracted with 80 mmol/L KCl. Thus, evidence from tissue, cellular, and molecular studies all implicates the BK_Ca channel as the effector molecule that mediates estrogen’s effect on coronary arteries. Subsequent experiments were designed to characterize the cellular mechanisms mediating this response.

View larger version (25K): [in this window] [in a new window]   Figure 3. Relaxation of coronary arteries by estrogen stimulation of BKCa channel activity. A, Continuous 500-millisecond traces from the same cell-attached patch at +40 mV before (control) and after 10 µmol/L 17ß-estradiol (40 minutes). Channel openings are upward deflections, and the dashed line indicates the closed state. B, BKCa channel activity is plotted as the probability of finding any channel in the open state during a 200-millisecond step to +40 mV in a cell-attached patch. Steps were repeated at 30-second intervals, and the open probabilities are plotted as vertical bars versus time after the beginning of the recording. Exposure to 10 µmol/L 17-estradiol or 10 µmol/L 17ß-estradiol is indicated by the bar. C, Contractile response of an endothelium-denuded coronary artery to 10 nmol/L iberiotoxin (IBTX). Arrow indicates point at which 10 µmol/L 17ß-estradiol (17ß-E) was added. The broken line indicates baseline tension.

Treating human coronary arteries with 3 µmol/L 17ß-estradiol nearly doubles cGMP content after 30 minutes, with a lesser increase reported for cAMP.¹¹ Interestingly, both cGMP and cAMP dilate porcine coronary arteries by activating the cGMP-dependent protein kinase (PKG).¹⁸ If estrogen-induced coronary vasodilation were mediated through the cGMP/PKG transduction process, then the effects of estrogen should be mimicked by application of exogenous cGMP. Treating coronary myocytes with 1 mmol/L 8-bromo-cGMP increased single BK_Ca channel NPo from .008 to .488 (n=3; Fig 4A) and also increased steady state outward currents in perforated-patch experiments by >100% (n=3, data not shown). Furthermore, the effect of 8-bromo-cGMP on BK_Ca channel gating was reversed when PKG activity was inhibited with either 100 µmol/L Rp-8-CPT-cGMPS (n=2, Fig 4A) or 300 nmol/L KT5823 (n=2, data not shown). Moreover, stimulating PKG activity with 100 µmol/L Sp-8-CPT-cGMPS enhanced BK_Ca channel NPo from .00 to .28 (Fig 4B). Previous studies with purified PKG have reported similar effects on BK_Ca channels in coronary smooth muscle¹⁹ and other cells.^{20 21} In addition, BK_Ca channel activity was stimulated when dephosphorylation was blocked with 500 nmol/L okadaic acid, a potent inhibitor of type I and II serine/threonine phosphoprotein phosphatases (Fig 4C). On average, okadaic acid increased BK_Ca channel NPo from .0002±.0002 to 4055±.2 (n=6) in cell-attached patches and also increased steady state outward currents by >100% (n=3; data not shown). Thus, it is apparent that cGMP-dependent phosphorylation stimulates BK_Ca channel activity in coronary arteries. Furthermore, inhibiting PKG activity with 300 nmol/L KT5823 (Fig 4D) produced a complete (99.4±0.8%; n=4) reversal of the stimulatory effect of 17ß-estradiol on BK_Ca channel NPo (control, .001; 17ß-estradiol, .183; KT5823, .003). Conversely, pretreating cells with 300 nmol/L KT5823 blocked the effect of 17ß-estradiol (n=3). Control experiments revealed that 1 µmol/L KT5823 had no effect on okadaic acid–stimulated channel activity (n=3) nor did it affect BK_Ca channels directly (n=3 inside-out patches). In addition, inhibiting PKG activity with 100 µmol/L Rp-8-CPT-cGMPS also blocked the effect of 17ß-estradiol (n=2 of 4).

View larger version (28K): [in this window] [in a new window]   Figure 4. Estrogen stimulation of BKCa channel activity via cGMP-dependent phosphorylation. Each panel represents a plot of channel activity from a different cell-attached patch during 100-millisecond intervals at +40 mV (holding potential, -60 mV). To conserve space on the computer’s hard disk, recording was interrupted at the times indicated by the broken lines on the time axis. Period of drug exposure is indicated by the bars. A, BKCa channel activity was stimulated by 1 mmol/L 8-bromo-cGMP (30-minute exposure), whereas 100 µmol/L Rp-8-CPT-cGMPS (20 minutes) reversed the effect. B, 100 µmol/L Sp-8-CPT-cGMPS (30 minutes) increased BKCa channel gating. C, 500 nmol/L okadaic acid (20 minutes) stimulated BKCa channel activity. D, BKCa channel activity was stimulated by a 20-minute exposure to 10 µmol/L 17ß-estradiol, and 300 nmol/L KT5823 reversed this stimulation after 7 minutes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is becoming increasingly apparent that many effects of sex steroids can be attributed to their vasoactive properties. The stimulatory effects of estrogen on uterine and uteroplacental blood flow are well known, and common manifestations of menopause-induced estrogen deficiency, eg, cessation of menses and vaginal dryness, are related directly to diminished blood flow. Moreover, many of these symptoms are largely alleviated when blood flow is restored by estrogen replacement therapy. Estrogen also enhances blood flow in nonreproductive tissues such as the skin, thyroid, and heart.^{9 22} Furthermore, significant decreases in systemic vascular resistance and diastolic blood pressure occur when estrogen levels are increased in humans^{6 7} or animals.¹⁶ Interestingly, sublingual administration of 17ß-estradiol has been used successfully to treat migraine attacks, suggesting a vasoactive effect of estrogen on cerebral vessels.⁸ Despite the potent ability of estrogen to control blood flow to a variety of organs and systems, the cellular or molecular basis of estrogen action on vascular smooth muscle remains undefined. The present study provides compelling evidence for a novel mechanism of estrogen-induced vasodilation: stimulation of an intracellular signaling mechanism, cGMP-dependent phosphorylation, that activates a specific effector protein, the BK_Ca channel. Opening of these channels would lead to K⁺ efflux, membrane repolarization, and relaxation of blood vessels.

Like other vascular smooth muscle cells, myocytes from coronary arteries possess substantial outward K⁺ currents.¹⁵ In the present study K⁺ currents were recorded from metabolically intact coronary myocytes with the perforated-patch technique.¹⁴ In addition to providing stable currents with minimal rundown, this technique obviates the need for calcium chelators, which are required for standard whole-cell recording. Thus, Ca²⁺-dependent processes, such as activation of ion channels, may be suppressed during whole-cell dialysis, but these mechanisms remain functional during perforated-patch recordings. Use of this technique revealed a large outward current that was composed mainly of BK_Ca channel activity. Subsequent single-channel experiments confirmed that BK_Ca channels dominated the electrical activity of membrane patches. Other studies have also demonstrated that BK_Ca channels are the predominant K⁺ channel species in coronary smooth muscle.¹⁵ Because of their large conductance (100 to 150 pS in physiological gradients of K⁺) and high density of expression, these channels help set and maintain the resting membrane potential of porcine coronary arteries,²³ and our experiments demonstrating iberiotoxin-induced contraction underscore the importance of BK_Ca channels in regulating arterial tone under nonstimulated conditions (Fig 3C). In addition, BK_Ca channels provide an important repolarizing negative-feedback mechanism to reverse active contraction as intracellular calcium increases. Given the importance of BK_Ca channels in regulating vascular tone, these channels would be an ideal effector molecule mediating the vasodilatory effect of 17ß-estradiol. This hypothesis is supported by the present findings that 17ß-estradiol stimulates BK_Ca channel gating in coronary myocytes and that estrogen was much less effective at relaxing arteries contracted with iberiotoxin, which blocks BK_Ca channels exclusively.¹⁷ Alternatively, vasodilation would also be produced if estrogen inhibited voltage-dependent calcium channels directly, which has been suggested in uterine arteries.²⁴ A recent study indicates that 17ß-estradiol increases Ca²⁺ current density in pituitary tumor cells,²⁵ but in this study 17-estradiol, a stereoisomer with far less biological activity,²⁶ was equally effective. Thus, the effects of estrogen on Ca²⁺ currents require further study. In the present study, however, blocking K⁺ efflux through BK_Ca channels with iberiotoxin or high extracellular [K⁺] depressed estrogen-induced relaxation by >80%. Although a portion of this residual relaxation might reflect direct inhibition of calcium channels, it is clear that the primary effect of estrogen on coronary arteries involves BK_Ca channels. Furthermore, the effect of 17ß-estradiol on BK_Ca channels was not due to a nonspecific, steroidal effect on plasma membrane fluidity or channel proteins, because 17-estradiol had no effect on BK_Ca channel gating at the cellular or molecular level (Figs 2 and 3). These experiments, combined with those demonstrating that PKG antagonists abolish the effect of 17ß-estradiol, strongly suggest that estrogen stimulates cGMP-dependent phosphorylation of the BK_Ca channel or a closely associated regulatory molecule.

Although the present data represent convincing evidence for a molecular mechanism that could account for estrogen’s ability to relax vascular smooth muscle in vivo, interpretation of these results requires an additional caveat. We observed that nanomolar amounts of 17ß-estradiol stimulated BK_Ca currents (Fig 2C); however, low-micromolar concentrations were routinely used to produce consistent, maximal responses. Thus, the majority of data was obtained with concentrations of estrogen far greater than the high picomolar-nanomolar levels of free hormone found in the plasma under normal (nonpregnant) conditions. It has generally been assumed that only free hormone is available for diffusion into target tissues; however, simple diffusion of these low levels cannot account completely for estrogen’s biological activity.²⁷ Moreover, the concentration of cellular exchangeable hormone necessary to cause 50% saturation of nuclear steroid receptors is 1 to 2 log orders greater than the concentration of unbound hormone.^{28 29} These and other studies argue for the existence of an additional pool of circulating steroid hormone that augments the amount available to target cells. This pool consists of steroid bound to plasma albumin or sex hormone binding globulin (SHBG) and comprises >90% of total plasma estrogen.³⁰ There is evidence that steroid hormone rapidly dissociates from plasma proteins during a single passage through the capillary microenvironment, whereas plasma proteins remain within the vascular space.^{28 29 31} Therefore, protein-bound hormone can function in vivo as a free fraction so that the actual concentration of capillary exchangeable hormone is in reality many times greater than the plasma concentration of free hormone.^{29 32} For example, target cells take up corticosterone or thyroxine from the albumin-bound plasma pool, and estradiol bound to albumin or SHBG is readily available for transcapillary diffusion into a number of tissues (eg, brain, liver, salivary gland, and uterine smooth muscle).²⁹ In light of these findings, free circulating levels of estrogen are probably a substantial underestimate of the actual capillary exchangeable pool. Experimental physiological salt solutions do not contain steroid binding proteins; thus, higher concentrations of free hormone are typically required in vitro to more closely simulate actual physiological conditions. Nonetheless, until the complicated process of steroid transport from blood into target cells is more completely understood, making a direct correlation between in vitro experimental data and in vivo conditions will remain somewhat problematic.

There is often a delay in the response of blood vessels to estrogen, and this response latency has been used as an indicator of genomic versus nongenomic mechanisms of action. For example, it requires 120 minutes for 17ß-estradiol to decrease systemic vascular resistance maximally in ewes,¹⁶ and this latency might reflect DNA transcription of a vasodilator compound. In other cells the response to estrogen is nongenomic in nature. 17ß-Estradiol stimulates release of Ca²⁺_i in granulosa cells after only 5 seconds.³³ In the present study, the effect of estrogen on intact arteries or on single cells often required 30 to 60 minutes to reach a maximum level, a response latency suggesting a nongenomic mechanism.¹¹ On the other hand, estrogen increases uterine blood flow within 20 to 30 minutes; however, this response was blocked when protein synthesis was inhibited with cycloheximide,³⁴ suggesting activation of DNA transcription during this comparatively short latency. In addition, previous studies have shown that RNA synthesis is stimulated by aldosterone after a latency of only 75 minutes in toad smooth muscle.³⁵ One explanation for these discrepancies might be found in a recent study demonstrating that [³H]17ß-estradiol incorporation into porcine LAD coronary arteries requires 30 to 45 minutes to reach equilibrium, with localization in both the nucleus and cytoplasm.³⁶ Thus, response latency may not be a reliable indicator of 17ß-estradiol’s mechanism of action in coronary arteries. In contrast, there is increasingly reliable evidence that production of cyclic nucleotides may mediate many of estrogen’s effects.

Recent studies indicate that estradiol stimulates cGMP and cAMP accumulation in human coronary arteries¹¹ and porcine ovarian cells.³⁷ Furthermore, 17ß-estradiol or nitroprusside inhibits thymidine uptake into porcine LAD coronary artery segments, suggesting involvement of cyclic nucleotides in estrogen’s antiproliferative effect.³⁶ In the present study the effect of 17ß-estradiol was mimicked by exogenous cGMP, activation of PKG, or phosphatase inhibition and was inhibited by PKG antagonists. These physiological studies, coupled with previous biochemical findings, strongly suggest that cGMP-dependent phosphorylation mediates the vascular effects of estrogen. An obvious mechanism of activating this transduction process would be stimulation of guanylyl cyclase activity; however, we are unaware of any studies demonstrating a direct effect of 17ß-estradiol on guanylyl cyclase. One possibility is that estrogen stimulates synthesis of a protein that activates the PKG transduction cascade. Interestingly, estradiol increases cytokine mRNA synthesis in uterine smooth muscle and bone.^{3 38} In arterial smooth muscle, interleukin 1, tumor necrosis factor-, or interferon- potentiates the activity of the cytokine-inducible nitric oxide synthase and increases cellular mRNA coding for this enzyme.^{39 40} Nitric oxide is a potent activator of soluble guanylyl cyclase in a number of cells, including vascular smooth muscle. Thus, it is tempting to speculate that estrogen elevates cGMP levels in vascular myocytes via nitric oxide. This mechanism could account for the vasodilator effect of estrogen in various vascular beds and the decreased peripheral resistance and diastolic pressure that occur during pregnancy⁶ and in estrogen-treated male transsexuals⁷ ; however, effects on nitric oxide synthase might require greater latency periods than those observed in the present study. Alternatively, other mechanisms (eg, inhibition of phosphodiesterase activity) might also play a role, and we have not ruled out the possibility that cAMP may also be involved in the cellular response to estrogen. Regardless, the present study links estrogen-stimulated cGMP-dependent phosphorylation with a membrane effector protein, the BK_Ca channel, which mediates relaxation of coronary arteries through a mechanism that was anticipated over 15 years ago.¹⁰ This vasodilatory mechanism might contribute to the lower incidence of coronary heart disease in premenopausal women and the efficacy of estrogen replacement therapy to reverse many of the cardiovascular complications of menopause. In light of the importance of cGMP-dependent phosphorylation in regulating smooth muscle tone and the abundance of BK_Ca channels expressed in smooth muscle and other excitable cells, these findings should also provide important insights into the cellular basis of estrogen’s effects on a number of tissues.

	Acknowledgments

 
Supported in part by the American Heart Association, Ohio Affiliate, Columbus, Ohio, the American Federation for Aging Research, and the Office of Geriatric Medicine and Gerontology and The Research Challenge Foundation (Wright State University). We are grateful for the technical assistance of K. Daugherty. We thank Drs R. Koerker and R. Grubbs for lending us the equipment to measure arterial contraction, and Drs L. Lu, P. Lauf, H.C. Hartzell, and D. Armstrong for their valuable comments concerning the manuscript. We also acknowledge the kind cooperation of Landes Meats and Focke’s Meats.

Received December 30, 1994; accepted July 13, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Kannel WB, Thom TJ. Incidence, prevalence and mortality of cardiovascular disease. In: Schlant RC, Alexander RW, eds. The Heart. New York, NY: McGraw-Hill, Inc; 1994:185-197.

2. Messerli FH, Garavaglia GE, Schmieder RE, Sundgaard-Riise K, Nunez BD, Amodeo C. Disparate cardiovascular findings in men and women with essential hypertension. Ann Intern Med.. 1987;107:158-161.

3. Sarrel PM, Lufkin EG, Oursler MJ, Keefe D. Estrogen actions in arteries, bone, and brain. Sci Am Sci Med.. 1994;1:44-53.

4. Webber LS, Hunter SM, Baugh JG, Srinivasan SR, Sklov MC, Berenson GS. The interaction of cigarette smoking, oral contraceptive use, and cardiovascular risk factor variables in children: the Bogalusa Heart Study. Am J Public Health.. 1982;72:266-274. [Abstract/Free Full Text]

5. Stampfer MJ, Colditz GA. Estrogen replacement therapy and coronary heart disease: a quantitative assessment of the epidemiologic evidence. Prev Med.. 1991;20:47-63. [Medline] [Order article via Infotrieve]

6. Elkayam U. Pregnancy and cardiovascular disease. In: Braunwald E, ed. Heart Disease. Philadelphia, Pa: W.B. Saunders; 1992:1790-1809.

7. Slater AJ, Gude N, Clarke IJ, Walters WAW. Haemodynamic changes and left ventricular performance during high-dose oestrogen administration to male transsexuals. Br J Obstet Gynaecol.. 1986;93:532-538. [Medline] [Order article via Infotrieve]

8. Sarrel PM. Ovarian hormones and the circulation. Maturitas.. 1990;590:287-298.

9. Raddino R, Manca C, Poli E, Bolognesi R, Visioli O. Effects of 17ß-estradiol on the isolated rabbit heart. Arch Int Pharmacodyn Ther.. 1986;281:57-65. [Medline] [Order article via Infotrieve]

10. Harder DR, Coulson PB. Estrogen receptors and effects of estrogen on membrane electrical properties of coronary vascular smooth muscle. J Cell Physiol.. 1979;100:375-382. [Medline] [Order article via Infotrieve]

11. Mugge A, Riedel M, Barton, Kuhn M, Lichtlen PR. Endothelium independent relaxation of human coronary arteries by 17ß-oestradiol in vitro. Cardiovasc Res.. 1993;27:1939-1942. [Medline] [Order article via Infotrieve]

12. White RE, Carrier GO. Vascular contraction induced by activation of membrane calcium ion channels is enhanced in streptozotocin-diabetes. J Pharmacol Exp Ther.. 1990;253:1057-1062. [Abstract/Free Full Text]

13. Clapp LH, Gurney AM. Outward currents in rabbit pulmonary artery cells dissociated with a new technique. Exp Physiol.. 1991;76:677-693.[Abstract]

14. Horn R, Marty A. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol.. 1988;92:145-159. [Abstract/Free Full Text]

15. Toro L, Scornik F. Modulation of Ca-activated K channels from coronary smooth muscle. In: Sperelakis N, Kuriyama H, eds. Ion Channels of Vascular Smooth Muscle Cells and Endothelial Cells. New York, NY: Elsevier; 1991:111-124.

16. Magness RR, Rosenfeld CR. Local and systemic estradiol-17ß: effects on uterine and systemic vasodilation. Am J Physiol.. 1989;256:E536-E542. [Abstract/Free Full Text]

17. Galvez A, Gimenez-Gallego G, Reuben JP, Roy-Contancin L, Feigenbaum P, Kaczorowski GJ, Garcia ML. Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus. J Biol Chem.. 1990;265:1083-1090.

18. Jiang H, Colbran JL, Francis SH, Corbin JD. Direct evidence for cross-activation of cGMP-dependent protein kinase by cAMP in pig coronary arteries. J Biol Chem.. 1992;267:1015-1019. [Abstract/Free Full Text]

19. Taniguchi S, Furukawa K, Munekazu S. Maxi K channels are stimulated by cyclic guanosine monophosphate-dependent protein kinase in canine coronary artery smooth muscle cells. Pflugers Arch.. 1993;423:167-172. [Medline] [Order article via Infotrieve]

20. White RE, Lee AB, Shcherbatko AD, Lincoln TM, Schonbrunn A, Armstrong DL. Potassium channel stimulation by natriuretic peptides through cGMP-dependent dephosphorylation. Nature.. 1993;361:263-266. [Medline] [Order article via Infotrieve]

21. Robertson BE, Schubert R, Hescheler J, Nelson MT. cGMP-dependent protein kinase activates Ca-activated K channels in cerebral artery smooth muscle cells. Am J Physiol.. 1993;265:C299-C303.[Abstract/Free Full Text]

22. Rosenfeld CR, Morriss FH, Battaglia FC, Makowski E, Meschia G. Effect of estradiol-17ß on blood flow to reproductive and nonreproductive tissues in pregnant ewes. Am J Obstet Gynecol.. 1976;124:618-629. [Medline] [Order article via Infotrieve]

23. Trieschmann U, Isenberg G. Ca²⁺-activated K channels contribute to the resting potential of vascular myocytes: Ca-sensitivity is increased by intracellular Mg-ions. Pflugers Arch.. 1989;414:S183-S184.

24. Stice SL, Ford SP, Rosazza JP, Van Order DE. Role of 4-hydroxylated estradiol in reducing Ca uptake of uterine arterial smooth muscle cells through potential-sensitive channels. Biol Reprod.. 1987;36:361-368. [Abstract]

25. Ritchie AK. Estrogen increases low voltage-activated calcium current density in GH₃ anterior pituitary cells. Endocrinology.. 1993;132:1621-1629. [Abstract/Free Full Text]

26. Guy CV, Kief NL, Bekker PJ. Effect of estrogen on acidification in osteoclasts. Biochem Biophys Res Commun.. 1993;192:1251-1259. [Medline] [Order article via Infotrieve]

27. Siiter PK, Nurai JT, Hammond GL, Nisker JA, Raymoure WJ, Kuhn RW. The serum transport of steroid hormones. Recent Prog Horm Res.. 1982;38:457-510.

28. Pardridge WM, Premachandra BN, Fierer G. Transport of thyroxine bound to human prealbumin into rat liver. Am J Physiol.. 1985;248:G545-G550.

29. Pardridge WM. Plasma protein-mediated transport of steroid and thyroid hormones. Am J Physiol.. 1987;252:E157-E164. [Abstract/Free Full Text]

30. Hsueh AJ, Billig H. Ovarian hormone synthesis and mechanism of action. In: Degroot LS, ed. Endocrinology. Philadelphia, Pa: W.B. Saunders Co; 1995;2019-2030.

31. Pardridge WM, Landaw EM. Testosterone transport in brain: primary role of plasma protein-bound hormone. Am J Physiol.. 1985;249:E534-E542. [Abstract/Free Full Text]

32. Wilson JD. Endocrinology. In: Isselbacher KJ, Braunwald E, Wilson JD, Martin JB, Fauci AS, Kasper DL, eds. Principles of Internal Medicine. New York, NY: McGraw-Hill, Inc; 1994:1884-1891.

33. Morley P, Whitfield JF, Vanderhyden BC, Tsang BK, Schwartz J. A new, nongenomic estrogen action: the rapid release of intracellular calcium. Endocrinology.. 1992;131:1305-1312. [Abstract/Free Full Text]

34. Killam AP, Rosenfeld CR, Battaglia FC, Makowski EL, Meschia G. Effect of estrogens on the uterine blood flow of oophorectomized ewes. Am J Obstet Gynecol.. 1973;115:1045-1052. [Medline] [Order article via Infotrieve]

35. Lahav M, Dietz T, Edelman IS. The action of aldosterone on sodium transport: further studies with inhibitors of RNA and protein synthesis. Endocrinology.. 1973;92:1685-1699. [Abstract/Free Full Text]

36. Vargas R, Wroblewska B, Rego A, Hatch J, Ramwell PW. Oestradiol inhibits smooth muscle cell proliferation of pig coronary artery. Br J Pharmacol.. 1993;109:612-617. [Medline] [Order article via Infotrieve]

37. Sirotkin AV, Nitray J. Steroid hormones regulate cAMP and cGMP production by porcine granulosa cells in vitro. J Steroid Biochem Mol Biol.. 1993;46:573-577.

38. Sahlin L, Norstedt G, Eriksson H. Estrogen regulation of the estrogen receptor and insulin-like growth factor-I in the rat uterus: a potential coupling between effects of estrogen and IGF-I. Steroids.. 1994;59:421-430. [Medline] [Order article via Infotrieve]

39. Beasley D, Schwartz JH, Brenner BM. Interleukin 1 induces prolonged L-arginine-dependent cyclic GMP and nitrite production in rat vascular smooth muscle cells. J Clin Invest.. 1991;87:602-608.

40. Pomerantz KB, Hajjar DP, Levi R, Gross S. Cholesterol enrichment of arterial smooth muscle cells upregulates cytokine-induced nitric oxide synthesis. Biochem Biophys Res Commun.. 1993;191:103-109.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				S. Zhu, D. D. Browning, R. E. White, D. Fulton, and S. A. Barman Mutation of protein kinase C phosphorylation site S1076 on {alpha}-subunits affects BKCa channel activity in HEK-293 cells Am J Physiol Lung Cell Mol Physiol, October 1, 2009; 297(4): L758 - L766. [Abstract] [Full Text] [PDF]

				G. Han, H. Ma, R. Chintala, D. J. R. Fulton, S. A. Barman, and R. E. White Essential Role of the 90-Kilodalton Heat Shock Protein in Mediating Nongenomic Estrogen Signaling in Coronary Artery Smooth Muscle J. Pharmacol. Exp. Ther., June 1, 2009; 329(3): 850 - 855. [Abstract] [Full Text] [PDF]

				C. R. Rosenfeld, X.-t. Liu, and K. DeSpain Pregnancy modifies the large conductance Ca2+-activated K+ channel and cGMP-dependent signaling pathway in uterine vascular smooth muscle Am J Physiol Heart Circ Physiol, June 1, 2009; 296(6): H1878 - H1887. [Abstract] [Full Text] [PDF]

				M. E. J. Lott, C. Hogeman, M. Herr, M. Bhagat, and L. I. Sinoway Sex differences in limb vasoconstriction responses to increases in transmural pressures Am J Physiol Heart Circ Physiol, January 1, 2009; 296(1): H186 - H194. [Abstract] [Full Text] [PDF]

				C. R. Rosenfeld, R. A. Word, K. DeSpain, and X.-t. Liu Large Conductance Ca2+--Activated K+ Channels Contribute to Vascular Function in Nonpregnant Human Uterine Arteries Reproductive Sciences, September 1, 2008; 15(7): 651 - 660. [Abstract] [PDF]

				Shu Zhu, R. E. White, and S. A. Barman Original Research: Role of phosphodiesterases in modulation of BKCa channels in hypertensive pulmonary arterial smooth muscle Therapeutic Advances in Respiratory Disease, June 1, 2008; 2(3): 119 - 127. [Abstract] [PDF]

				V. M. Miller and S. P. Duckles Vascular Actions of Estrogens: Functional Implications Pharmacol. Rev., June 1, 2008; 60(2): 210 - 241. [Abstract] [Full Text] [PDF]

				P.-A. Scott, A. Tremblay, M. Brochu, and J. St-Louis Vasorelaxant action of 17 -estradiol in rat uterine arteries: role of nitric oxide synthases and estrogen receptors Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3713 - H3719. [Abstract] [Full Text] [PDF]

				G. Han, H. Ma, R. Chintala, K. Miyake, D. J. R. Fulton, S. A. Barman, and R. E. White Nongenomic, endothelium-independent effects of estrogen on human coronary smooth muscle are mediated by type I (neuronal) NOS and PI3-kinase-Akt signaling Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H314 - H321. [Abstract] [Full Text] [PDF]

				T. Traupe, C. D. Stettler, H. Li, E. Haas, I. Bhattacharya, R. Minotti, and M. Barton Distinct Roles of Estrogen Receptors {alpha} and {beta} Mediating Acute Vasodilation of Epicardial Coronary Arteries Hypertension, June 1, 2007; 49(6): 1364 - 1370. [Abstract] [Full Text] [PDF]

				A. Moritz, R. Gust, and H. H. Pertz Characterization of the Relaxant Response to N,N'-Dipropyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine in Porcine Coronary Arteries J. Pharmacol. Exp. Ther., May 1, 2007; 321(2): 699 - 706. [Abstract] [Full Text] [PDF]

				C. Castillo, G. Ceballos, D. Rodriguez, C. Villanueva, R. Medina, J. Lopez, E. Mendez, and E. F. Castillo Effects of estradiol on phenylephrine contractility associated with intracellular calcium release in rat aorta Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1388 - C1394. [Abstract] [Full Text] [PDF]

				J. Titze, F. C. Luft, K. Bauer, P. Dietsch, R. Lang, R. Veelken, H. Wagner, K.-U. Eckardt, and K. F. Hilgers Extrarenal Na+ Balance, Volume, and Blood Pressure Homeostasis in Intact and Ovariectomized Deoxycorticosterone-Acetate Salt Rats Hypertension, June 1, 2006; 47(6): 1101 - 1107. [Abstract] [Full Text] [PDF]

				C. M. Masi, L. C. Hawkley, J. D. Berry, and J. T. Cacioppo Estrogen Metabolites and Systolic Blood Pressure in a Population-Based Sample of Postmenopausal Women J. Clin. Endocrinol. Metab., March 1, 2006; 91(3): 1015 - 1020. [Abstract] [Full Text] [PDF]

				G. Han, X. Yu, L. Lu, S. Li, H. Ma, S. Zhu, X. Cui, and R. E. White Estrogen Receptor {alpha} Mediates Acute Potassium Channel Stimulation in Human Coronary Artery Smooth Muscle Cells J. Pharmacol. Exp. Ther., March 1, 2006; 316(3): 1025 - 1030. [Abstract] [Full Text] [PDF]

				R. E. White, G. Han, C. Dimitropoulou, S. Zhu, K. Miyake, D. Fulton, S. Dave, and S. A. Barman Estrogen-induced contraction of coronary arteries is mediated by superoxide generated in vascular smooth muscle Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1468 - H1475. [Abstract] [Full Text] [PDF]

				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]

				C. W. Leffler, A. L. Fedinec, H. Parfenova, and J. H. Jaggar Permissive contributions of NO and prostacyclin in CO-induced cerebrovascular dilation in piglets Am J Physiol Heart Circ Physiol, July 1, 2005; 289(1): H432 - H438. [Abstract] [Full Text] [PDF]

				C. Dimitropoulou, R. E. White, D. R. Ownby, and J. D. Catravas Estrogen Reduces Carbachol-Induced Constriction of Asthmatic Airways by Stimulating Large-Conductance Voltage and Calcium-Dependent Potassium Channels Am. J. Respir. Cell Mol. Biol., March 1, 2005; 32(3): 239 - 247. [Abstract] [Full Text] [PDF]

				J. Brouillette, K. Rivard, E. Lizotte, and C. Fiset Sex and strain differences in adult mouse cardiac repolarization: importance of androgens Cardiovasc Res, January 1, 2005; 65(1): 148 - 157. [Abstract] [Full Text] [PDF]

				D. K. Bowles, K. K. Maddali, V. K. Ganjam, L. J. Rubin, D. L. Tharp, J. R. Turk, and C. L. Heaps Endogenous testosterone increases L-type Ca2+ channel expression in porcine coronary smooth muscle Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2091 - H2098. [Abstract] [Full Text] [PDF]

				K. Kurata, M. Takebayashi, S. Morinobu, and S. Yamawaki {beta}-Estradiol, Dehydroepiandrosterone, and Dehydroepiandrosterone Sulfate Protect against N-Methyl-D-aspartate-Induced Neurotoxicity in Rat Hippocampal Neurons by Different Mechanisms J. Pharmacol. Exp. Ther., October 1, 2004; 311(1): 237 - 245. [Abstract] [Full Text] [PDF]

				S. Ling, J.-Z. Sheng, and A. P. Braun The calcium-dependent activity of large-conductance, calcium-activated K+ channels is enhanced by Pyk2- and Hck-induced tyrosine phosphorylation Am J Physiol Cell Physiol, September 1, 2004; 287(3): C698 - C706. [Abstract] [Full Text] [PDF]

				S. A. Barman, S. Zhu, and R. E. White PKC activates BKCa channels in rat pulmonary arterial smooth muscle via cGMP-dependent protein kinase Am J Physiol Lung Cell Mol Physiol, June 1, 2004; 286(6): L1275 - L1281. [Abstract] [Full Text] [PDF]

				J. Palacios, E. T. Marusic, N. C. Lopez, M. Gonzalez, and L. Michea Estradiol-induced expression of Na+-K+-ATPase catalytic isoforms in rat arteries: gender differences in activity mediated by nitric oxide donors Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1793 - H1800. [Abstract] [Full Text] [PDF]

				K. H. Mikulec, L. Holloway, R. E. Krasnow, H. Javitz, G. E. Swan, T. Reed, R. Marcus, and D. Carmelli Relationship of Endogenous Sex Hormones to Coronary Heart Disease: A Twin Study J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1240 - 1245. [Abstract] [Full Text] [PDF]

				J. M. Orshal and R. A. Khalil Gender, sex hormones, and vascular tone Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2004; 286(2): R233 - R249. [Abstract] [Full Text] [PDF]

				F. L. Wynne, J. A. Payne, A. E. Cain, J. F. Reckelhoff, and R. A. Khalil Age-Related Reduction in Estrogen Receptor-Mediated Mechanisms of Vascular Relaxation in Female Spontaneously Hypertensive Rats Hypertension, February 1, 2004; 43(2): 405 - 412. [Abstract] [Full Text] [PDF]

				S. A. Barman, S. Zhu, and R. E. White Protein kinase C inhibits BKCa channel activity in pulmonary arterial smooth muscle Am J Physiol Lung Cell Mol Physiol, January 1, 2004; 286(1): L149 - L155. [Abstract] [Full Text] [PDF]

				M. E. DiSanto Corpus Cavernosum Smooth Muscle Physiology: A Role for Sex Hormones? J Androl, November 1, 2003; 24(6_suppl): S6 - S16. [Full Text] [PDF]

				R. Chatrath, K. L. Ronningen, P. LaBreche, S. R. Severson, M. Jayachandran, M. P. Bracamonte, and V. M. Miller Effect of puberty on coronary arteries from female pigs J Appl Physiol, October 1, 2003; 95(4): 1672 - 1680. [Abstract] [Full Text] [PDF]

				M. C. Rodrigo, D. S. Martin, and K. M. Eyster Estrogen decreases biglycan mRNA expression in resistance blood vessels Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2003; 285(4): R754 - R761. [Abstract] [Full Text] [PDF]

				M. I. Bahamonde, J. M. Fernandez-Fernandez, F. X. Guix, E. Vazquez, and M. A. Valverde Plasma Membrane Voltage-dependent Anion Channel Mediates Antiestrogen-activated Maxi Cl- Currents in C1300 Neuroblastoma Cells J. Biol. Chem., August 29, 2003; 278(35): 33284 - 33289. [Abstract] [Full Text] [PDF]

				S. A. Barman, S. Zhu, G. Han, and R. E. White cAMP activates BKCa channels in pulmonary arterial smooth muscle via cGMP-dependent protein kinase Am J Physiol Lung Cell Mol Physiol, June 1, 2003; 284(6): L1004 - L1011. [Abstract] [Full Text] [PDF]

				C. R. Rosenfeld, C. Chen, T. Roy, and X.-T. Liu Estrogen Selectively Up-Regulates eNOS and nNOS in Reproductive Arteries By Transcriptional Mechanisms Reproductive Sciences, May 1, 2003; 10(4): 205 - 215. [Abstract] [PDF]

				T. Simoncini, E. Rabkin, and J. K. Liao Molecular Basis of Cell Membrane Estrogen Receptor Interaction With Phosphatidylinositol 3-Kinase in Endothelial Cells Arterioscler Thromb Vasc Biol, February 1, 2003; 23(2): 198 - 203. [Abstract] [Full Text] [PDF]

				K. J. Ho and J. K. Liao Nonnuclear Actions of Estrogen Arterioscler Thromb Vasc Biol, December 1, 2002; 22(12): 1952 - 1961. [Abstract] [Full Text] [PDF]

				M. P. Bracamonte, M. Jayachandran, K. S. Rud, and V. M. Miller Acute effects of 17beta -estradiol on femoral veins from adult gonadally intact and ovariectomized female pigs Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2389 - H2396. [Abstract] [Full Text] [PDF]

				Y. Ito, S. Sato, M. Son, M. Kondo, H. Kume, K. Takagi, and K. Yamaki Bisphenol A Inhibits Cl- Secretion by Inhibition of Basolateral K+ Conductance in Human Airway Epithelial Cells J. Pharmacol. Exp. Ther., July 1, 2002; 302(1): 80 - 87. [Abstract] [Full Text] [PDF]

				K. J. Ho and J. K. Liao Non-nuclear Actions of Estrogen: New Targets for Prevention and Treatment of Cardiovascular Disease Mol. Interv., July 1, 2002; 2(4): 219 - 228. [Abstract] [Full Text] [PDF]

				M. A. Sader and D. S. Celermajer Endothelial function, vascular reactivity and gender differences in the cardiovascular system Cardiovasc Res, February 15, 2002; 53(3): 597 - 604. [Full Text] [PDF]

				T. S Mikkola and T. B Clarkson Estrogen replacement therapy, atherosclerosis, and vascular function Cardiovasc Res, February 15, 2002; 53(3): 605 - 619. [Abstract] [Full Text] [PDF]

				R. E White, G. Han, M. Maunz, C. Dimitropoulou, A. M El-Mowafy, R. S Barlow, J. D Catravas, C. Snead, G. O Carrier, S. Zhu, et al. Endothelium-independent effect of estrogen on Ca2+-activated K+ channels in human coronary artery smooth muscle cells Cardiovasc Res, February 15, 2002; 53(3): 650 - 661. [Abstract] [Full Text] [PDF]

				R. K. Dubey, S. Oparil, B. Imthurn, and E. K. Jackson Sex hormones and hypertension Cardiovasc Res, February 15, 2002; 53(3): 688 - 708. [Abstract] [Full Text] [PDF]

				F. A Babiker, L. J De Windt, M. van Eickels, C. Grohe, R. Meyer, and P. A Doevendans Estrogenic hormone action in the heart: regulatory network and function Cardiovasc Res, February 15, 2002; 53(3): 709 - 719. [Abstract] [Full Text] [PDF]

				C. J. Gruber, W. Tschugguel, C. Schneeberger, and J. C. Huber Production and Actions of Estrogens N. Engl. J. Med., January 31, 2002; 346(5): 340 - 352. [Full Text] [PDF]

				A. Q. Ding and J. N. Stallone Testosterone-induced relaxation of rat aorta is androgen structure specific and involves K+ channel activation J Appl Physiol, December 1, 2001; 91(6): 2742 - 2750. [Abstract] [Full Text] [PDF]

				D. A. Lewis, M. P. Bracamonte, K. S. Rud, and V. M. Miller Genome and Hormones: Gender Differences in Physiology: Selected Contribution: Effects of sex and ovariectomy on responses to platelets in porcine femoral veins J Appl Physiol, December 1, 2001; 91(6): 2823 - 2830. [Abstract] [Full Text] [PDF]

				A. W. Miller, C. Dimitropoulou, G. Han, R. E. White, D. W. Busija, and G. O. Carrier Epoxyeicosatrienoic acid-induced relaxation is impaired in insulin resistance Am J Physiol Heart Circ Physiol, October 1, 2001; 281(4): H1524 - H1531. [Abstract] [Full Text] [PDF]

				V. P. Deenadayalu, R. E. White, J. N. Stallone, X. Gao, and A. J. Garcia Testosterone relaxes coronary arteries by opening the large-conductance, calcium-activated potassium channel Am J Physiol Heart Circ Physiol, October 1, 2001; 281(4): H1720 - H1727. [Abstract] [Full Text] [PDF]

				J. R. Rouleau, A. Dagnault, D. Simard, B. Lavallee, A. Belanger, A. Blouin, and J. G. Kingma Jr. Effect of estrogen replacement therapy on distribution of myocardial blood flow in female anesthetized rabbits Am J Physiol Heart Circ Physiol, September 1, 2001; 281(3): H1407 - H1412. [Abstract] [Full Text] [PDF]

				Y. Watanabe, M. T. Littleton-Kearney, R. J. Traystman, and P. D. Hurn Estrogen restores postischemic pial microvascular dilation Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H155 - H160. [Abstract] [Full Text] [PDF]

				C. R. Rosenfeld, D. N. Cornfield, and T. Roy Ca2+-activated K+ channels modulate basal and E2{beta}-induced rises in uterine blood flow in ovine pregnancy Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H422 - H431. [Abstract] [Full Text] [PDF]

				R. K. Dubey and E. K. Jackson Estrogen-induced cardiorenal protection: potential cellular, biochemical, and molecular mechanisms Am J Physiol Renal Physiol, March 1, 2001; 280(3): F365 - F388. [Abstract] [Full Text] [PDF]

				C. Dimitropoulou, R. E. White, L. Fuchs, H. Zhang, J. D. Catravas, and G. O. Carrier Angiotensin II Relaxes Microvessels Via the AT2 Receptor and Ca2+-Activated K+ (BKCa) Channels Hypertension, February 1, 2001; 37(2): 301 - 307. [Abstract] [Full Text] [PDF]

				A. Federici, M. Ciccone, E. Cicinelli, M. Lombardi, M. V. Pitzalis, P. Galantino, and V. Pinto Effects of Estrogen Therapy on Arterial Resistance and Pulsatility in Postmenopausal Women Angiology, January 1, 2001; 52(1): 15 - 23. [Abstract] [PDF]

				A. K. Singh, B. D. Schultz, J. A. Katzenellenbogen, E. M. Price, R. J. Bridges, and N. A. Bradbury Estrogen Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator-Mediated Chloride Secretion J. Pharmacol. Exp. Ther., October 1, 2000; 295(1): 195 - 204. [Abstract] [Full Text]

				R. S. Barlow, A. M. El-Mowafy, and R. E. White H2O2 opens BKCa channels via the PLA2-arachidonic acid signaling cascade in coronary artery smooth muscle Am J Physiol Heart Circ Physiol, August 1, 2000; 279(2): H475 - H483. [Abstract] [Full Text] [PDF]

				G. G. Geary, D. N. Krause, and S. P. Duckles Estrogen reduces mouse cerebral artery tone through endothelial NOS- and cyclooxygenase-dependent mechanisms Am J Physiol Heart Circ Physiol, August 1, 2000; 279(2): H511 - H519. [Abstract] [Full Text] [PDF]

				C. R. Rosenfeld, R. E. White, T. Roy, and B. E. Cox Calcium-activated potassium channels and nitric oxide coregulate estrogen-induced vasodilation Am J Physiol Heart Circ Physiol, July 1, 2000; 279(1): H319 - H328. [Abstract] [Full Text] [PDF]

				W. A. Salhab, P. W. Shaul, B. E. Cox, and C. R. Rosenfeld Regulation of types I and III NOS in ovine uterine arteries by daily and acute estrogen exposure Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H2134 - H2142. [Abstract] [Full Text] [PDF]

				R. E. White, J. P. Kryman, A. M. El-Mowafy, G. Han, and G. O. Carrier cAMP-Dependent Vasodilators Cross-Activate the cGMP-Dependent Protein Kinase to Stimulate BKCa Channel Activity in Coronary Artery Smooth Muscle Cells Circ. Res., April 28, 2000; 86(8): 897 - 905. [Abstract] [Full Text] [PDF]

				N. C. Adragna, R. E. White, S. N. Orlov, and P. K. Lauf K-Cl cotransport in vascular smooth muscle and erythrocytes: possible implication in vasodilation Am J Physiol Cell Physiol, February 1, 2000; 278(2): C381 - C390. [Abstract] [Full Text] [PDF]

				A. B Ropero, E. Fuentes, J. M Rovira, C. Ripoll, B. Soria, and A. Nadal Non-genomic actions of 17{beta}-oestradiol in mouse pancreatic {beta}-cells are mediated by a cGMP-dependent protein kinase J. Physiol., December 1, 1999; 521(2): 397 - 407. [Abstract] [Full Text] [PDF]

				M. A. Valverde, P. Rojas, J. Amigo, D. Cosmelli, P. Orio, M. I. Bahamonde, G. E. Mann, C. Vergara, and R. Latorre Acute Activation of Maxi-K Channels (hSlo) by Estradiol Binding to the  Subunit Science, September 17, 1999; 285(5435): 1929 - 1931. [Abstract] [Full Text]

				S. Tanabe, T. Hata, and M. Hiraoka Effects of estrogen on action potential and membrane currents in guinea pig ventricular myocytes Am J Physiol Heart Circ Physiol, August 1, 1999; 277(2): H826 - H833. [Abstract] [Full Text] [PDF]

				M. E. Mendelsohn and R. H. Karas The Protective Effects of Estrogen on the Cardiovascular System N. Engl. J. Med., June 10, 1999; 340(23): 1801 - 1811. [Full Text] [PDF]

				V M Miller Gender and vascular reactivity Lupus, June 1, 1999; 8(5): 409 - 415. [Abstract] [PDF]

				R. Schubert, T. Noack, and V. N. Serebryakov Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells Am J Physiol Cell Physiol, March 1, 1999; 276(3): C648 - C658. [Abstract] [Full Text] [PDF]

				Y. S. Prakash, A. A. Togaibayeva, M. S. Kannan, V. M. Miller, L. A. Fitzpatrick, and G. C. Sieck Estrogen increases Ca2+ efflux from female porcine coronary arterial smooth muscle Am J Physiol Heart Circ Physiol, March 1, 1999; 276(3): H926 - H934. [Abstract] [Full Text] [PDF]

				R. J. Gonzales and N. L. Kanagy Endothelium-Independent Relaxation of Vascular Smooth Muscle by 17{beta}-Estradiol Journal of Cardiovascular Pharmacology and Therapeutics, January 1, 1999; 4(4): 227 - 234. [Abstract] [PDF]

				R. S. Barlow and R. E. White Hydrogen peroxide relaxes porcine coronary arteries by stimulating BKCa channel activity Am J Physiol Heart Circ Physiol, October 1, 1998; 275(4): H1283 - H1289. [Abstract] [Full Text] [PDF]

				T. J. K. Toung, R. J. Traystman, P. D. Hurn, and V. M. Miller Estrogen-Mediated Neuroprotection After Experimental Stroke in Male Rats • Editorial Comment Stroke, August 1, 1998; 29(8): 1666 - 1670. [Abstract] [Full Text] [PDF]

				M. Kahonen, J.-P. Tolvanen, K. Sallinen, X. Wu, and I. Porsti Influence of gender on control of arterial tone in experimental hypertension Am J Physiol Heart Circ Physiol, July 1, 1998; 275(1): H15 - H22. [Abstract] [Full Text] [PDF]

				G. G. Geary, D. N. Krause, and S. P. Duckles Estrogen reduces myogenic tone through a nitric oxide-dependent mechanism in rat cerebral arteries Am J Physiol Heart Circ Physiol, July 1, 1998; 275(1): H292 - H300. [Abstract] [Full Text] [PDF]

				M. Nara, P. D. K. Dhulipala, Y.-X. Wang, and M. I. Kotlikoff Reconstitution of beta -Adrenergic Modulation of Large Conductance, Calcium-activated Potassium (Maxi-K) Channels in Xenopus Oocytes. IDENTIFICATION OF THE cAMP-DEPENDENT PROTEIN KINASE PHOSPHORYLATION SITE J. Biol. Chem., June 12, 1998; 273(24): 14920 - 14924. [Abstract] [Full Text] [PDF]

				D. O. Ruehlmann, J. R. Steinert, M. A. Valverde, R. Jacob, and G. E. Mann Environmental estrogenic pollutants induce acute vascular relaxation by inhibiting L-type Ca2+ channels in smooth muscle cells FASEB J, May 1, 1998; 12(7): 613 - 619. [Abstract] [Full Text]

				N. J. Alkayed, I. Harukuni, A. S. Kimes, E. D. London, R. J. Traystman, P. D. Hurn, and P. A. Grady Gender-Linked Brain Injury in Experimental Stroke • Editorial Comment Stroke, January 1, 1998; 29(1): 159 - 166. [Abstract] [Full Text] [PDF]

				D. F. Skafar, R. Xu, J. Morales, J. Ram, and J. R. Sowers Female Sex Hormones and Cardiovascular Disease in Women J. Clin. Endocrinol. Metab., December 1, 1997; 82(12): 3913 - 3918. [Abstract] [Full Text] [PDF]

				D. B. Dixon and D. R. Copenhagen Metabotropic Glutamate Receptor-Mediated Suppression of an Inward Rectifier Current Is Linked via a cGMP Cascade. J. Neurosci., December 1, 1997; 17(23): 8945 - 8954. [Abstract] [Full Text] [PDF]

				G. M.C. Rosano, A. M. Caixeta, S. Chierchia, S. Arie, M. Lopez-Hidalgo, W. I. Pereira, F. Leonardo, C. M. Webb, F. Pileggi, and P. Collins Short-term Anti-Ischemic Effect of 17ß-Estradiol in Postmenopausal Women With Coronary Artery Disease Circulation, November 4, 1997; 96(9): 2837 - 2841. [Abstract] [Full Text]

				K. A. Roberts, M. M. Woo, and J. C. Rutledge Nitric Oxide Mediates LDL Uptake in the Artery Wall in Response to High Concentrations of 17ß-Estradiol Arterioscler Thromb Vasc Biol, October 1, 1997; 17(10): 2123 - 2131. [Abstract] [Full Text]

				R.-S. Zhang, P. H. Guth, O. U. Scremin, R. Singh, S. Pervin, and G. Chaudhuri Regulation of endometrial blood flow in ovariectomized rats: assessment of the role of nitric oxide Am J Physiol Heart Circ Physiol, October 1, 1997; 273(4): H2009 - H2017. [Abstract] [Full Text] [PDF]

				K. Node, M. Kitakaze, H. Kosaka, T. Minamino, H. Funaya, and M. Hori Amelioration of Ischemia- and Reperfusion-Induced Myocardial Injury by 17ß-Estradiol : Role of Nitric Oxide and Calcium-Activated Potassium Channels Circulation, September 16, 1997; 96(6): 1953 - 1963. [Abstract] [Full Text]

				E. Gilad, H. Matzkin, and N. Zisapel Interplay between Sex Steroids and Melatonin in Regulation of Human Benign Prostate Epithelial Cell Growth J. Clin. Endocrinol. Metab., August 1, 1997; 82(8): 2535 - 2541. [Abstract] [Full Text] [PDF]

				B. D. Johnson, W. Zheng, K. S. Korach, T. Scheuer, W. A. Catterall, and G. M. Rubanyi Increased Expression of the Cardiac L-type Calcium Channel in Estrogen Receptor-deficient Mice J. Gen. Physiol., August 1, 1997; 110(2): 135 - 140. [Abstract] [Full Text] [PDF]

				K. D. Dunlap, M. L. McAnelly, and H. H. Zakon Estrogen Modifies an Electrocommunication Signal by Altering the Electrocyte Sodium Current in an Electric Fish, Sternopygus J. Neurosci., April 15, 1997; 17(8): 2869 - 2875. [Abstract] [Full Text] [PDF]

				N. Bowling, W. E. Bloomquist, M. L. Cohen, H. U. Bryant, H. W. Cole, D. E. Magee, E. R. Rowley, and C. J. Vlahos Effects of Prolonged Ethinyl Estradiol Treatment on Calcium Channel Binding and In Vivo Calcium-Mediated Hemodynamic Responses in Ovariectomized Rats J. Pharmacol. Exp. Ther., April 1, 1997; 281(1): 218 - 225. [Abstract] [Full Text]

				G. C. Wellman, A. D. Bonev, M. T. Nelson, and J. E. Brayden Gender Differences in Coronary Artery Diameter Involve Estrogen, Nitric Oxide, and Ca2+-Dependent K+ Channels Circ. Res., November 1, 1996; 79(5): 1024 - 1030. [Abstract] [Full Text]

				K. J. Scheidegger, B. Cenni, D. Picard, and P. Delafontaine Estradiol Decreases IGF-1 and IGF-1 Receptor Expression in Rat Aortic Smooth Muscle Cells. MECHANISMS FOR ITS ATHEROPROTECTIVE EFFECTS J. Biol. Chem., December 1, 2000; 275(49): 38921 - 38928. [Abstract] [Full Text] [PDF]

				M. Di Fulvio, T. M. Lincoln, P. K. Lauf, and N. C. Adragna Protein Kinase G Regulates Potassium Chloride Cotransporter-3 Expression in Primary Cultures of Rat Vascular Smooth Muscle Cells J. Biol. Chem., June 8, 2001; 276(24): 21046 - 21052. [Abstract] [Full Text] [PDF]

				M. Jankowski, G. Rachelska, W. Donghao, S. M. McCann, and J. Gutkowska Estrogen receptors activate atrial natriuretic peptide in the rat heart PNAS, September 25, 2001; 98(20): 11765 - 11770. [Abstract] [Full Text] [PDF]

				C. Dimitropoulou, G. Han, A. W. Miller, M. Molero, L. C. Fuchs, R. E. White, and G. O. Carrier Potassium (BKCa) currents are reduced in microvascular smooth muscle cells from insulin-resistant rats Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H908 - H917. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by White, R. E. Articles by Falvo Lang, J. L. Search for Related Content PubMed PubMed Citation Articles by White, R. E. Articles by Falvo Lang, J. L.