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(Circulation Research. 1996;79:1024-1030.)
© 1996 American Heart Association, Inc.

Articles

Gender Differences in Coronary Artery Diameter Involve Estrogen, Nitric Oxide, and Ca²⁺-Dependent K⁺ Channels

George C. Wellman, Adrian D. Bonev, Mark T. Nelson, Joseph E. Brayden

the Department of Pharmacology, The University of Vermont, UVM Medical Research Facility, Colchester.

Correspondence to Dr Mark T. Nelson, Department of Pharmacology, The University of Vermont, UVM Medical Research Facility, 55A South Park Dr, Colchester, VT 05446. E-mail nelson@northpole.med.uvm.edu.

	Abstract

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During their reproductive years, women have a much lower incidence of coronary heart disease compared with men of similar age. Estrogen appears to be largely responsible for this decrease in cardiovascular mortality in women. In the present study, isolated pressurized coronary arteries from rats were used to assess the role of gender and circulating estrogen on coronary vascular function. Pressure-induced constrictions ("myogenic tone") were greater (2-fold) in isolated coronary arteries from estrogen-deficient male or ovariectomized (OVX) rats compared with similar arteries obtained from female rats or OVX rats receiving physiological levels of estrogen replacement (OVX+E group). These differences in coronary artery diameter were abolished by removal of the vascular endothelium or chemical inhibition of NO synthase. The anti-estrogen, tamoxifen, increased pressure-induced constrictions of coronary arteries from female and OVX+E rats. Dilations of pressurized coronary arteries from female and OVX animals to sodium nitroprusside, a nitrovasodilator that generates NO, were reduced by >50% by iberiotoxin (IBTX), an inhibitor of Ca²⁺-dependent K⁺ (K_Ca) channels. Sodium nitroprusside (10 µmol/L) hyperpolarized coronary arteries by 13±2 mV, an effect that was greatly diminished (80%) by IBTX. Coronary arteries isolated from female rats produced greater constrictions in response to IBTX and KT 5823, an inhibitor of cGMP-dependent protein kinase, compared with coronary arteries from OVX rats. cGMP-dependent protein kinase increased the activity of K_Ca channels 16.5±5-fold in excised membrane patches from smooth muscle cells enzymatically isolated from these small coronary arteries. We propose that physiological levels of circulating 17ß-estradiol elevate basal NO release from the endothelial cells, which increases the diameter of pressurized coronary arteries. Further, our results suggest that part of the effect of this NO is through activation of K_Ca channels in the smooth muscle cells of the coronary arteries.

Key Words: coronary artery • estrogen • nitric oxide • K⁺ channels • vasodilation

	Introduction

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Women experience a dramatic increase in the incidence of coronary heart disease when estrogen levels drop during menopause. Physiological levels of estrogen (17ß-estradiol) clearly exert a cardioprotective effect, with postmenopausal women being two to three times less likely to develop heart disease if receiving estrogen replacement therapy.^{1 2} Favorable alterations in serum lipids and decreased vascular reactivity are both thought to contribute to the cardioprotective effect of estrogen.^{3 4 5} However, the precise pathways and mechanisms by which estrogen influences coronary arteries are unclear.

Recent evidence suggests that NO production may play an important role in the effects of estrogen on the vasculature. A positive correlation has been found between plasma 17ß-estradiol and levels of stable metabolites of NO during follicular development in women.⁶ Consistent with a role for NO, endothelium-dependent coronary artery vasodilation is increased after 17ß-estradiol treatment in ovariectomized monkeys⁷ and postmenopausal women.⁸ Endothelium-dependent relaxation of conduit arteries (aorta, femoral artery) in vitro is also enhanced by estrogen.^{9 10} Basal NO production by endothelial cells may also be related to plasma estrogen status. Inhibition of NO synthase produces a greater increase in tension in partially contracted aortic segments from female rabbits compared with male or OVX rabbits.¹¹ Enhanced production of NO in rat aortas from females versus males has been observed using a bioassay system,¹² and induction of both the neuronal and endothelial isozymes of NO synthase is increased in estrogen-treated animals.¹³

Although there are clear indications that endothelial function is altered by estrogen, the effects of endogenous estrogen on intrinsic or myogenic tone of coronary arteries have not been elucidated. To understand the effect of estrogen on coronary artery diameter, we studied small (200 µm in diameter) coronary arteries that constrict to physiological levels of intravascular pressure.¹⁴ This maintained constriction ("myogenic tone") regulates blood flow to target organs, including the heart.¹⁵ In the present study, we report the first demonstration of gender differences and estrogen effects on myogenic tone of coronary arteries. Our data show that endogenous 17ß-estradiol decreases myogenic tone through enhanced tonic release of the vasodilator NO by endothelial cells. Further, we provide evidence for a pathway involving activation of K_Ca channels in the smooth muscle cells by G-kinase, which links the estrogen-induced increase in NO release to vasodilation.

	Materials and Methods

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Animals and Tissues
Four groups of Sprague-Dawley rats (16 weeks of age) were used in the present study: male, normal cycling female (referred to as the female group), and ovariectomized rats with and without physiological levels of estrogen replacement (OVX and OVX+E groups, respectively). Ovariectomy was performed in 9-week-old rats by the supplier (Charles River, St. Constant, Canada). For estrogen replacement, ovariectomized animals were given a subcutaneous timed release 17ß-estradiol pellet (60-day release, 0.25 mg per pellet, Innovative Research of America) 3 days after ovariectomy and were studied 7 weeks later. Some of the OVX, OVX+E, and normal female animals were treated with tamoxifen (10 mg/kg per week IP) for a period of 6 weeks before study. Rats were euthanized with an overdose of pentobarbital, and blood was collected for later measurement of plasma 17ß-estradiol by radioimmunoassay (DPC 17ß-estradiol kit, ICN Biomedicals, Inc). The heart was then removed and placed in cold Krebs' bicarbonate buffer (mmol/L: NaCl 119, KCl 3, KH₂PO₄ 1.7, MgSO₄ 1.2, NaHCO₃ 25, EDTA 0.02, CaCl₂ 1.6, and glucose 11, pH 7.4). The right ventricle was then opened along the free wall, and segments of the septal artery (200 µm in diameter, 2 to 3 mm in length) were removed.

Diameter and Membrane Potential Measurements
Isolated coronary arteries were studied using a myograph chamber (Living Systems Instruments) as previously described.^{16 17} The arteries were continuously superfused with warmed (37°C) Krebs' buffer, and diameter was monitored using a video dimension analyzer system. At the end of each experiment, passive properties (pressure-diameter relationship and distension ratio) of the arteries were determined after a 10-minute exposure to Ca²⁺-free Krebs' buffer, which eliminates active force development in this tissue.¹⁸ Removal of the endothelial lining was accomplished by passing air through the lumen of the cannulated artery for 2 minutes, followed by perfusion with 1 mL of distilled water. Removal of the endothelium was confirmed by the loss of acetylcholine-induced dilations. Membrane potential measurements were recorded using conventional intracellular microelectrodes in isometrically mounted rat coronary arteries depolarized with LNNA and prostaglandin F₂ to physiological membrane potentials (-40 to -45 mV).¹⁹

Patch-Clamp Studies
Single K_Ca channel currents were recorded from "inside-out" membrane patches using the patch-clamp technique.²⁰ Septal artery smooth muscle cells were enzymatically isolated as described previously.²¹ Segments of coronary artery were incubated at 37°C in a HEPES buffer (mmol/L: NaCl 60, sodium glutamate 80, KCl 5, MgCl₂ 2, glucose 10, HEPES 10, and CaCl₂ 0.1, along with 1 mg/mL bovine serum albumin) containing 1 mg/mL papain and 1 mg/mL dithioerythritol for 20 minutes. Arteries were then incubated in a similar HEPES buffer containing collagenase (type F, 2 mg/mL) and hyaluronidase (1 mg/mL) for 15 minutes. Arterial segments were then washed in cold HEPES buffer, and single myocytes were dispersed by trituration. Isolated smooth muscle cells were transferred to a recording chamber containing a solution of the following composition (mmol/L): KCl 130, MgCl₂ 1, HEPES 10, EGTA 3, and CaCl₂ 2.808, pH 7.4 (adjusted with KOH), along with 1 µmol/L free Ca²⁺; total K⁺ was 145 mmol/L. Patch pipettes were made from borosilicate glass (Sutter Instrument Co) and were filled with a solution containing (mmol/L): NaCl 120, KCl 20, MgCl₂ 1, HEPES 10, and CaCl₂ 0.1, pH 7.4. Electrode resistances ranged from 3 to 7 M. Single-channel currents were measured using an Axopatch 200A amplifier (Axon Instruments) and recorded on a DAT recorder (DTC-700, Sony). Data were filtered at 1 kHz using an eight-pole Bessel filter and digitized at 5 kHz. Average channel activity (NP_o) was determined from 10-minute recordings as follows: NP_o=_j=1^Nt_j/T, where P_o is the open-state probability, T is the duration of the recording, t_j is the time spent with j=1,2,...N channels open, and N is the maximal number of channels observed.

Macroscopic K_Ca channel currents were recorded using the perforated-patch whole-cell recording technique as previously described.²¹ The cells were bathed in a solution of the following composition (mmol/L): NaCl 134, KCl 6, MgCl₂ 1, HEPES 10, CaCl₂ 2.0, and glucose 10, pH 7.4. The patch pipette contained (mmol/L) potassium aspartate 110, KCl 30, NaCl 10, HEPES 10, MgCl₂ 1_, and EGTA 0.05, pH 7.2, along with 200 µg/mL amphotericin. Cells were held at a membrane potential of -50 mV. Step depolarizations to +40 mV were applied for a period of 500 milliseconds, first in the absence of estrogen and then in the same cell after 30 minutes of exposure to 5 µmol/L 17ß-estradiol.

Drugs
The following agents were obtained from Sigma Chemical Co unless otherwise noted: acetylcholine, adenosine triphosphate, L-arginine, 17ß-estradiol (Innovative Research of America), G-kinase (Promega Chemical), cGMP, HEPES, IBTX (Peptides International), KT 5823 (Calbiochem), LNNA, prostaglandin F_2, SNP, and tamoxifen citrate.

Statistical Analysis
Data are expressed as mean±SEM; n indicates the number of animals. Comparisons of data between two groups were made using Student's t test. Data among three or more groups were compared using ANOVA, and post hoc comparisons of individual groups were made using the Newman-Keuls test. Data were considered to be significantly different at values of P<.05.

	Results

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Over the physiological range of intravascular pressures (40 to 100 mm Hg), coronary arteries isolated from female rats were significantly less constricted than corresponding arteries from male rats (Fig 1A and 1B). This gender difference in coronary artery diameter appears to be due to circulating levels of endogenous 17ß-estradiol (female plasma 17ß-estradiol: 21±4 pg/mL [77 pmol/L], n=22; male plasma 17ß-estradiol: <5 pg/mL [<18 pmol/L], n=9), because coronary arteries isolated from OVX rats (plasma 17ß-estradiol: <5 pg/mL [<18 pmol/L], n=8) had diameters similar to arteries from male rats (Fig 1B). Furthermore, replacing circulating 17ß-estradiol (to 45±10 pg/mL [165 pmol/L], n=8) in the OVX+E animals by implanting time-released 17ß-estradiol pellets for 6 weeks increased the diameter of isolated arteries to the level observed for arteries isolated from untreated female animals (Fig 1B). In contrast to the marked differences in myogenic tone, the passive mechanical properties (pressure-diameter relationships in Ca²⁺-free buffer, Fig 1A; pressure-distension ratio relationships, data not shown) of the arteries from female, male, OVX, and OVX+E groups were similar, suggesting no differences in the structural composition of the arterial wall.⁸ Therefore, physiological levels of 17ß-estradiol appear to be responsible for the increased arterial diameter of pressurized coronary arteries from female animals. Coronary arteries were never exposed to exogenous estrogen after removal from the animals, yet differences in arterial tone between female (or OVX+E) and male (or OVX) persisted for the entire duration of experiments (6 to 10 hours), consistent with a genomic effect of 17ß-estradiol.²²

View larger version (25K): [in this window] [in a new window]   Figure 1. Female coronary arteries are less constricted than male arteries. A, Increases in intravascular pressure produced a greater reduction in diameter (constriction) in arteries from male rats compared with arteries from female rats (solid tracings), with no observed differences in the passive properties of the arterial wall (dotted lines). B, Arteries from estrogen-deficient female rats (OVX, n=5) and male rats (n=5) constricted more (P<.01, ANOVA) to intravascular pressures (40 mm Hg) than arteries from cycling females (n=8) and 17ß-estradiol–treated ovariectomized rats (OVX+E, n=5). C, Physical removal of the endothelial lining of these coronary arteries abolished differences in constrictions between treatment groups (n=3 or 4). D, Chemical inhibition of the enzyme NO synthase by incubation of arteries with LNNA (50 µmol/L) increased pressure-induced tone. After LNNA treatment, constrictions were not different in arteries from females and males or in arteries from the OVX and OVX+E groups (not shown). These data also demonstrate that the effects of LNNA are greater in arteries from females compared with males, suggesting an enhanced release of NO from female arteries.

The possibility that estrogen was acting on the endothelium to cause a tonic vasodilation was explored, since estrogen had been reported to enhance receptor-mediated endothelium-dependent relaxations⁹ or increase basal NO release¹¹ and NO synthase expression¹² in large arteries. Indeed, we found that removal of the endothelium (Fig 1C) or exposure of arteries to an inhibitor of NO synthase (LNNA) (Fig 1D) abolished the differences in diameter of coronary arteries isolated from female, male, OVX, and OVX+E animals. Therefore, estrogen appears to increase the basal release of NO from the endothelial cells lining female coronary arteries. Differences in coronary artery diameter were not due to an enhanced sensitivity of female or OVX+E coronary arteries to NO, since these arteries were actually less sensitive than male or OVX arteries to SNP, a nitrovasodilator that generates NO and increases intracellular cGMP^{23 24} (SNP IC₅₀ values: males, 3.8x10^-8 mol/L; females, 1.6x10^-7 mol/L; OVX group, 7.9x10^-8 mol/L; and OVX+E group, 6.3x10^-7 mol/L). Other investigators have demonstrated an increased sensitivity of vascular smooth muscle to nitrates after blockade of NO synthase.²⁵ Therefore, the higher sensitivity to SNP in male and OVX arteries is consistent with the possibility that lower tonic release of NO occurs in arteries from male and OVX rats compared with female and OVX+E rats.

To examine further the involvement of the estrogen receptor in the decrease in pressure-induced constrictions in female and OVX+E animals, we treated animals with tamoxifen, an estrogen-receptor antagonist. Six weeks of treatment with tamoxifen increased the pressure-induced constrictions in female and OVX+E rats to levels observed for control OVX rats (Fig 2). This observation supports the hypothesis that the effects of estrogen are mediated via estrogen receptors, presumably located in the endothelial cells. Paradoxically, tamoxifen treatment of estrogen-deficient animals (OVX group) attenuated the constrictions to elevated intravascular pressure (Fig 2, OVX+tamoxifen versus OVX groups). This result suggests that in the absence of estrogen, tamoxifen has a weak estrogen-like effect.

View larger version (15K): [in this window] [in a new window]   Figure 2. Tamoxifen enhances pressure-induced constrictions in coronary arteries from female rats and ovariectomized rats treated with estrogen (FEM+TAM and EST+TAM, respectively). Constrictions of arteries from FEM+TAM rats (n=5) and EST+TAM rats (n=5) were larger over the pressure range of 60 to 100 mm Hg compared with ovariectomized rats treated with estrogen but not receiving tamoxifen (EST) (P<.05). Pressure-induced constrictions in arteries from EST and OVX+TAM animals were not significantly different. OVX indicates nontreated ovariectomized rats.

We also sought possible targets for NO. The exact mechanisms by which NO dilates coronary arteries are not known but presumably involve NO activation of guanylyl cyclase, an elevation of intracellular cGMP, and stimulation of G-kinase, which ultimately leads to vasodilation.²⁶ Recent evidence indicates that G-kinase can activate K_Ca channels in various types of smooth muscle,^{27 28 29 30 31} and this may contribute to vasodilation by closing voltage-dependent Ca²⁺ channels through membrane potential hyperpolarization.³² Therefore, we tested the hypothesis that NO acts in part to dilate coronary arteries through G-kinase–mediated activation of K_Ca channels in the coronary artery smooth muscle. In support of this hypothesis, IBTX (30 nmol/L), a specific blocker of K_Ca channels,³³ inhibited dilations of arteries from female and OVX rats to SNP (10 µmol/L) by 52±4% and 55±5%, respectively (Fig 3A). If SNP acts by opening K_Ca channels, then it should cause an IBTX-sensitive membrane potential hyperpolarization. Indeed, SNP (10 µmol/L) hyperpolarized the coronary artery vascular smooth muscle cells by 13±2 mV (n=7), an effect that was reduced by 85% after exposure to 30 nmol/L IBTX (Fig 3B). These findings indicate that nitrovasodilators act in part to dilate coronary arteries through stimulation of K_Ca channels.

View larger version (13K): [in this window] [in a new window]   Figure 3. The nitrovasodilator SNP (10 µmol/L) dilates and hyperpolarizes isolated rat myogenic arteries through activation of KCa channels. A, Diameter measurements obtained from OVX coronary arteries pressurized to 80 mm Hg and exposed to SNP under control conditions or after incubation with IBTX (30 nmol/L, which selectively inhibits KCa channels). SNP dilations were reduced by >50% (n=5) after IBTX (P<.01). Maximal dilations (expressed as percentage of the 0 Ca2+ response) to SNP were not significantly different in arteries from male (84±5%, n=7), female (81±3%, n=12), OVX (90±6%, n=4), or OVX+E (76±14%, n=3) rats. B, Hyperpolarizations to SNP (10 µmol/L) in coronary arteries in the absence and presence of IBTX. Mean SNP-induced hyperpolarization in control (n=7) and IBTX-treated (n=3) arteries (P<.01, IBTX vs control) are shown in the bar graphs to the right.

Our hypothesis would also predict that under conditions of enhanced tonic release of NO (eg, in female coronary arteries), activity of G-kinase and K_Ca channels should be elevated. Therefore, blockers of G-kinase (KT 5823³⁴ ) and K_Ca channels (IBTX) would be expected to cause a greater constriction (ie, reversal of NO-induced dilation) in coronary arteries from female compared with OVX rats. To compare the effects of the G-kinase inhibitor and IBTX on female and OVX arteries at the same initial diameters, OVX coronary arteries were dilated to the diameter of female coronary arteries using levcromakalim, an ATP-sensitive K⁺ channel opener.³² Under these conditions, differences in K_Ca activity should be caused primarily by external factors such as NO activity rather than by variations in intracellular Ca²⁺ associated with different levels of tone. KT 5823 (Fig 4A) and IBTX (Fig 4B) produced constrictions of 24±2% and 22±4%, respectively, of pressurized (80 mm Hg) coronary arteries from females. In contrast, KT 5823 and IBTX constricted pressurized coronary arteries from OVX animals by only 9±1% and 8±2%, respectively. These results are consistent with the idea that female coronary arteries have less myogenic tone than OVX or male coronary arteries, because enhanced NO release from female arteries leads to dilation, in part through activation of K_Ca channels.

View larger version (14K): [in this window] [in a new window]   Figure 4. Estrogen increases basal production of NO, as reflected in greater maximum constrictions (P<.01) of isolated pressurized (80 mm Hg) coronary arteries from female rats than OVX rats in response to inhibition of G-kinase with KT 5823 (1 µmol/L) (A) or inhibition of KCa channels with IBTX (30 nmol/L) (B).

Our pharmacological studies using intact coronary arteries suggest that G-kinase can enhance K_Ca channel activity in coronary artery myocytes. We provide further direct evidence for this mechanism by examining the effects of G-kinase on single K_Ca channels in inside-out excised patches from smooth muscle cells isolated from the same myogenic coronary arteries (Fig 5A and 5B). Agents were added to the cytoplasmic face of the membrane. These single-channel currents exhibited properties similar to that reported previously for Ca²⁺-dependent K⁺ channels³² (slope conductance=152.3±1.5 pS, n=7; removal of Ca²⁺ from the bathing solution inhibits channel activity, and membrane depolarization increases channel open-state probability). G-kinase (5 U/µL) increased the activity of single K_Ca channels by 16.5±6-fold (n=7). Channel activation by G-kinase was observed only in the presence of cGMP (100 µmol/L) and ATP (100 µmol/L), suggesting that channel phosphorylation is a necessary step in the activation cascade.

View larger version (16K): [in this window] [in a new window]   Figure 5. A, G-kinase–enhanced KCa channel activity. B, Summary of data showing that G-kinase in the presence of ATP and cGMP increased KCa activity 16.5±6.4-fold (P<.01) at a holding potential (Vh) of 0 mV. NPo indicates average channel activity, where Po is the open-state probability, and N is the maximal number of channels observed. C, Proposed model to explain the role of estrogen in reducing myogenic tone in coronary arteries.

We also tested the direct effects of estrogen on macroscopic K_Ca currents. In the absence of 17ß-estradiol, whole-cell K_Ca currents at +40 mV were 19.32±1.12 pA/pF; 30 minutes after exposure to 5 µmol/L 17ß-estradiol, K_Ca channel currents were 15.96±0.90 pA/pF (P<.05 versus control, n=4). Whole-cell currents returned to control values after removal of 17ß-estradiol from the bath.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we provide the first evidence that myogenic tone, a fundamental contractile mechanism in the resistance vasculature, is substantially lower in coronary arteries exposed in vivo to physiological levels of estrogen (female and OVX+E rats) compared with arteries exposed to little or no circulating estrogen (male and OVX rats). Our results are consistent with the following mechanism (described in two steps) for these gender- and estrogen-related differences in myogenic tone (see Fig 5C): (1) Physiological levels (50 to 100 pmol/L) of estrogen increase tonic NO release from the endothelial cells. This increase in NO release may involve elevated expression of NO synthase,¹³ although other mechanisms may be involved. (2) NO activates guanylyl cyclase, which increases cGMP and thereby stimulates cGMP-dependent protein kinase, leading to increased activity of K_Ca channels in the smooth muscle cells. Activation of K⁺ channels causes membrane hyperpolarization, which would close voltage-dependent Ca²⁺ channels, decrease intracellular Ca²⁺, and lead to vasodilation.¹⁶ It is clear that NO will also activate one or more complementary vasodilator pathways, including other cGMP-mediated effects, such as inhibition of Ca²⁺ channels,³⁵ enhanced Ca²⁺ extrusion, and changes in Ca²⁺ sensitivity³⁶ ; direct activation of K_Ca channels by NO has also been reported.³⁷ It should also be noted that in addition to enhancing the release of NO, which leads to greater activation of G-kinase, estrogen could enhance the ability of the G-kinase to activate K_Ca channels. However, the lower sensitivity of female coronary arteries to SNP (the present study) would argue against an additional action of estrogen on guanylyl cyclase, cGMP, or G-kinase activities. Further, cGMP may act in part through stimulation of cAMP-dependent protein kinase,³⁸ which can also activate K_Ca channels.³⁹ In addition to direct activation of K_Ca channels by G-kinase (Fig 5), this kinase or cAMP-dependent protein kinase could also elevate K_Ca channel activity through enhancing the amplitude or frequency of Ca²⁺-release events ("Ca²⁺ sparks") through ryanodine-sensitive Ca²⁺ channels in the subsarcolemmal sarcoplasmic reticulum. Ca²⁺ sparks appear to be a major regulator of K_Ca channel activity in myogenic arteries⁴⁰ and therefore may represent an additional synergistic pathway of NO-induced K_Ca channel activation.

A few cautionary statements concerning the above interpretations are warranted: (1) The rat was chosen as the animal model in the present study because the hormonal status of the animal can be altered relatively easily (ovariectomy and estrogen replacement), and viable, small, myogenic coronary arteries can be consistently isolated from the rat heart. The heart rate and metabolic demands of the heart in the rat are, however, significantly greater than in larger mammals. This could mean that mechanisms of regulation of the coronary circulation in the rat, including those related to myogenic tone, are dissimilar to those in other species. However, the characteristics of myogenic tone in the isolated rat coronary arteries (pressure-diameter relationships, dependence on extracellular calcium, and magnitude) are similar to those observed in other species,⁴¹ indicating that regulation of myogenic tone among different species and tissues may in fact be similar. Additional studies in other species are required to clarify this issue. (2) Measurements of diameter (pressurized arteries), membrane potential (isometric rings), and K_Ca channel activity (single cells) were made using a variety of techniques and could not be made simultaneously. We have assumed that the basic mechanisms of regulation of vascular tone are maintained in the various tissue configurations, which, of course, may or may not be correct. However, we are not aware of evidence to suggest that this assumption is incorrect. In addition, the data from each of the three approaches are internally consistent; eg, NO- and cGMP-mediated activation of vasodilation, hyperpolarization, and K⁺ channel activity are all inhibited by K_Ca channel blockers, suggesting a common cellular mechanism in each of the tissue configurations. (3) Ovariectomy will obviously alter the plasma levels of hormones and factors (progesterone and trophic hormones) other than estrogen, and such changes could also influence coronary artery diameter. Our studies do not rule out this possibility. However, estrogen replacement in OVX rats completely restores the "female" myogenic response (Fig 1C), arguing against a significant role of other factors that might be altered by ovariectomy. Therefore, we believe our interpretations and conclusions drawn from these studies are reasonable.

High concentrations (5 to 10 µmol/L) of exogenous estrogen (physiological levels are 10^-4 µmol/L) have been found to activate K_Ca channels in porcine coronary artery smooth muscle cells independently of any action on the endothelium.⁴² Inhibition of protein kinase G abolished this effect, suggesting that estrogen can activate G-kinase in the vascular smooth muscle cell via some unidentified pathway. The results of the study cited above are in sharp contrast with the present work, in which we have examined the effects of physiological levels of circulating estrogen (50 to 100 pmol/L) and have found that estrogen-induced changes in coronary artery tone mediated by K_Ca channels are absolutely dependent on the presence of an intact endothelium. Furthermore, we have tested the direct effects of high concentrations (5 µmol/L) of estrogen on K_Ca channel currents in isolated rat coronary artery myocytes and, unlike White et al,⁴² did not observe activation of K_Ca channel currents by estrogen in this preparation. Thus, additional studies are required to determine the physiological significance of possible direct actions of estrogen on K_Ca channels in vascular smooth muscle.

Tamoxifen is an estrogen receptor antagonist/partial agonist that is widely used in the adjuvant treatment of estrogen receptor–positive breast cancer. In the present study, treatment with tamoxifen increased pressure-induced constrictions in female and OVX+E animals, consistent with a reversal of the estrogen-induced increase in basal NO. In estrogen-deficient animals, however, tamoxifen treatment actually reduced pressure-induced constrictions of isolated coronary arteries. Such "estrogen-like" effects of tamoxifen in estrogen-deficient states have been observed by others. For instance, tamoxifen has been reported to decrease low-density lipoprotein and increase high-density lipoprotein plasma levels in postmenopausal women⁴³ and increase uterine and bone growth in ovariectomized rats.⁴⁴ Thus, the observations with tamoxifen in the present study are consistent with the hypothesis that estrogen-induced increases in basal NO production represent an estrogen receptor–mediated event. These results also imply that tamoxifen therapy in patients with normal estrogen status could conceivably interfere with the normal cardioprotective effect of estrogen in the coronary circulation.

An estrogen-induced increase in NO within the coronary circulation would provide several advantages. First, NO may decrease thrombosis and atherosclerosis by reducing platelet aggregation and adhesion to the vascular endothelium.⁴⁵ Second, a basal increase in NO may reduce the likelihood or severity of an ischemic event, since NO plays a major role in regulating coronary artery diameter and blood flow.⁴⁶ Third, membrane potential hyperpolarization reduces contractile responses to a variety of vasoconstrictors,^{47 48} suggesting that an NO-mediated hyperpolarization could lower the incidence of coronary artery spasm in response to vasoconstrictive stimuli. Understanding the mechanisms underlying the actions of estrogen on coronary arteries could aid in the development of new therapeutic strategies, by which cardioprotective effects might be separated from other components of estrogen activity, potentially decreasing the incidence of cardiovascular disease in both sexes.

	Selected Abbreviations and Acronyms

 

G-kinase = cGMP-dependent protein kinase KCa channel = Ca2+-dependent K+ channel IBTX = iberiotoxin LNNA = N-nitro-L-arginine OVX group = ovariectomized rats OVX+E group = rats ovariectomized and treated with estrogen SNP = sodium nitroprusside

	Acknowledgments

 
This study was supported by grants from the National Institutes of Health (HL-51728, HL-44455, and HL-35911) and an Established Investigator Award of the American Heart Association to Dr Brayden. We thank J. Patlak, H. Knot, V. Porter, and J. Jaggar for helpful discussions and comments. We also thank Andra Stevenson for his outstanding technical assistance with these studies.

Received February 22, 1996; accepted July 26, 1996.

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