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(Circulation. 1997;96:2795-2801.)
© 1997 American Heart Association, Inc.

Articles

The Role of Nitric Oxide in Coronary Vascular Effects of Estrogen in Postmenopausal Women

Victor Guetta, MD; Arshed A. Quyyumi, MD; Abhiram Prasad, MD; Julio A. Panza, MD; Myron Waclawiw, PhD; ; Richard O. Cannon, III, MD

From the Cardiology Branch and Office of Biostatistics Research, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md.

Correspondence to Dr Richard O. Cannon III, National Institutes of Health, Bldg 10, Room 7B15, 10 Center Dr MSC1650, Bethesda, MD 20892-1650.

	Abstract

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Background At physiological concentrations, 17ß-estradiol selectively enhances endothelium-dependent coronary vasodilation by an unknown mechanism in postmenopausal women.

Methods and Results To assess the contribution of nitric oxide (NO) to the vascular effects of estradiol, we measured coronary epicardial and microvascular responses to intracoronary acetylcholine (range, 3 to 300 µg/min for 2 minutes) before and after intracoronary estradiol 75 ng/min for 15 minutes in 20 estrogen-deficient women, 16 of whom had angiographic evidence of atherosclerosis or risk factors for atherosclerosis. This testing was repeated after inhibition of NO synthesis with intracoronary N^G-monomethyl-L-arginine (L-NMMA) 64 µmol/min for 5 minutes. Estradiol increased acetylcholine-stimulated coronary flow from 54±48% (mean±SD) above baseline values before estradiol infusion to 100±63% above baseline values (P=.007) and decreased coronary resistance from 32±21% to 46±15% below baseline values (P=.007) at a coronary sinus estradiol concentration of 1725±705 pmol/L (470±192 pg/mL). Estradiol also tended to lessen the severity of acetylcholine-induced epicardial coronary artery vasoconstriction from 8±11% to 3±11% below baseline values (P=.123). However, during L-NMMA infusion, estradiol no longer potentiated the effects of acetylcholine on coronary flow dynamics; coronary flow increased 39±46% above baseline values and coronary resistance decreased 19±30% below baseline values (both P<.001 versus pre–L-NMMA responses). The epicardial diameter decreased 8±11% below baseline values (P=.06 versus pre–L-NMMA response).

Conclusions The effects of estradiol at physiological concentrations on endothelium-dependent coronary vasodilator responsiveness in postmenopausal women are mediated by enhanced bioavailability of NO, which may be responsible in part for the cardioprotective effects of estrogen.

Key Words: atherosclerosis • coronary disease • nitric oxide • endothelium • hormones

	Introduction

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Cardiovascular disease due to atherosclerosis is the leading cause of morbidity and mortality in women living in developed societies, as it is for men, although the clinically apparent onset of disease expression is shifted by approximately a decade later in women. This delay in disease expression relative to men may in part be due to antiatherogenic effects of estrogen before failure of ovarian function during menopause. The Nurses' Health Study reported an 50% reduction in cardiovascular risk in postmenopausal women currently on estrogen therapy compared with women who had never used estrogens.¹ The beneficial effect of estrogen therapy in postmenopausal women may in part result from increases in HDL cholesterol and reduction in LDL cholesterol to a more favorable ratio, retarding atherogenesis,^{2 3 4} although epidemiological studies question whether alteration in lipid profile alone can account for all of the apparent cardiovascular benefit of estrogen therapy.⁵

Animal studies have indicated that estrogen may have vascular effects independent of changes in lipoprotein profile. Thus intravenous administration of 17ß-estradiol to ovariectomized primates fed an atherogenic diet was found to reverse acutely the epicardial coronary artery response to acetylcholine from constriction to dilation without any effect on nitroglycerin-mediated vasodilation,⁶ suggesting estrogen-mediated improvement in endothelial function. This study is consistent with the demonstration of estrogen-enhanced endothelium-dependent relaxation of rabbit femoral artery and swine coronary artery rings to acetylcholine.^{7 8} We found that estradiol infused intra-arterially to achieve physiological levels in postmenopausal women potentiated endothelium-dependent vasodilation in both the forearm⁹ and coronary circulations.¹⁰ Other groups have also shown that acute or chronic administration of estrogen to postmenopausal women improves endothelium-dependent coronary vasodilator function.^{11 12 13}

However, the mechanism of the endothelial effects of estradiol in these animal and human studies is unknown. Potentiation of acetylcholine-stimulated flow by estrogen could result from vascular smooth muscle relaxation caused by enhanced production or release of endothelium-derived relaxing factors such as nitric oxide (NO)^{14 15} and prostacyclin¹⁶ or inhibition of the release or activity of vasoconstrictor substances such as endothelin¹⁷ and angiotensin II.¹⁸ Because NO also has other potential endothelium-dependent antiatherogenic properties^{19 20} that could account for the cardioprotective effects of estrogen, as suggested by observational angiographic studies²¹ and epidemiological surveys,¹ we undertook the present study to determine whether the alteration of coronary vascular reactivity observed after administration of estrogen to postmenopausal women is mediated by enhanced bioavailability of NO.

	Methods

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Study Population
Twenty-five postmenopausal women (average age, 59 years; range, 44 to 77), all of whom had serum estradiol levels <184 pmol/L (50 pg/mL) and who were undergoing cardiac catheterization for evaluation of chest pain symptoms, were enrolled in this study. Twenty women had one or more risk factors for atherosclerosis: LDL cholesterol 4.14 mmol/L (160 mg/dL) (10 women), hypertension (10 women), diabetes mellitus (5 women), and current smoker (2 women). Eleven women had angiographic evidence of coronary atherosclerosis. None had received estrogen therapy within the preceding 2 months, only 1 had previously received lipid-lowering therapy, and all had discontinued aspirin and nonsteroidal anti-inflammatory agents for at least 2 weeks before entry into the study. The study was approved by the National Heart, Lung, and Blood Institute Investigational Review Board, and all study participants gave written informed consent.

Study Protocol
Cardiac catheterizations were performed after an overnight fast, with 10 mg diazepam given orally as premedication. Additional diazepam (2 to 3 mg) was given intravenously as needed to maintain patient comfort during the study. After angiography with 10 000 U heparin for anticoagulation, a 6F Judkins catheter was advanced to the ostium of the left or right coronary artery. A 0.018-in, 12-MHz Doppler wire (Cardiometrics Inc) was advanced through this catheter into the proximal coronary artery that was angiographically normal or with <50% stenosis: the left anterior descending artery in 19 women, the left circumflex artery in 4 women, and the left main and right coronary arteries in 1 woman each. The wire tip was positioned such that a characteristic and stable flow velocity waveform was obtained. Coronary angiograms were obtained in the right anterior oblique (for the left coronary artery) and in the left anterior oblique (for the right coronary artery) projections for measurement of epicardial coronary diameters at baseline and after each drug infusion, with hand injections of 5 mL ioxaglate (Mallinckrodt Medical). Quantitative measurements of coronary artery dimensions were made in the proximal coronary artery 0.5 cm distal to the wire tip using a computer-based edge enhancement technique (Quantim 2001, ImageComm Systems, Inc) by a technician who had no knowledge of the identity of the drugs infused before each of the angiograms. On-line measurements were made of average peak flow velocity, and each value was taken as the average of two cardiac cycles. A quantitative estimate of coronary blood flow was calculated from the Doppler flow velocity and quantitative angiographic data using the following equation:

where Q is flow (mL/min), D is vessel diameter (mm), and APV is average peak velocity (cm/s).²² Coronary resistance was derived as mean blood pressure÷coronary blood flow. In 14 women, a 6F multipurpose catheter was introduced through the right internal jugular vein into the mid coronary sinus to sample coronary venous blood for estradiol levels before and after estradiol infusions. Estradiol concentrations were subsequently measured by radioimmunoassay (Diagnostic Products Corp).

Study of Endothelium-Dependent Vasodilator Responsiveness
After basal measurement of coronary artery flow velocity and angiography during intracoronary infusion of 5% dextrose in water at 1 mL/min, acetylcholine (Sigma) dissolved in 5% dextrose in water solution was infused into the coronary artery for 2 minutes at a rate of 1 mL/min, at which time coronary flow velocity measurement and angiography were performed to assess endothelium-dependent vascular responsiveness. Acetylcholine was infused in the following concentrations: 3, 30, 100, and 300 µg/min, with each dose administered for 2 minutes. Escalating doses of acetylcholine were given until two doses were identified as producing the greatest increases in coronary flow velocity in the absence of fluoroscopically apparent epicardial constriction.

Estradiol Effect on Coronary Vasomotion
17ß-Estradiol (USP, Inc) in 2.5% ethanol diluent at a concentration of 75 ng/mL was infused through the left coronary catheter at 1 mL/min for 15 minutes in 22 women and at a concentration of 37.5 ng/mL at 1 mL/min for 15 minutes into the right coronary catheter in 1 woman and into infusion catheters positioned selectively in the left anterior descending and circumflex coronary arteries in 2 women. We have previously found that estradiol 75 ng/min infused into the left coronary artery achieves coronary venous estradiol plasma levels similar to those of reproductive-aged women at midcycle.^{10 23} The coronary flow velocity responses to the doses of acetylcholine that earlier produced the highest peak flow velocity responses before estradiol infusion were measured and angiography performed during continued infusion of estradiol.

Because two infusions of estradiol were required to investigate the contribution of NO to the vascular effects of estradiol (1 without and 1 with inhibition of NO synthesis), the first 5 women underwent sequential infusions of estradiol after a 30-minute rest to assess the reproducibility of the effect of estradiol on acetylcholine-stimulated coronary vascular dynamics. Coronary flow velocity, angiography, and coronary venous sampling for estradiol levels were performed before and after 15 minutes of each of the estradiol infusions.

Estradiol Effect After NO Inhibition
After a 30-minute rest period, the vasomotor effect of estradiol after inhibition of NO synthesis was assessed in the remaining 20 women who participated in this study. After basal measurements of coronary flow velocity and angiography, N^G-monomethyl-l-arginine (L-NMMA, Calbiochem Corp), an analogue of l-arginine that competitively inhibits NO synthesis,²⁴ was infused at a concentration of 64 µmol/mL into the coronary artery at 1 mL/min in 17 women, with measurements of coronary flow velocity and angiography after 5 minutes of infusion. The 3 women described above who received half-dose infusions of estradiol selectively into the right, left anterior descending, and circumflex coronary arteries also received half-doses of L-NMMA infused directly into the same respective arteries. Measurement of coronary flow velocity and angiography were then performed after reinfusion of the dose of acetylcholine that earlier produced the highest coronary flow velocity response during the infusion of estradiol. The L-NMMA infusion was then stopped, and estradiol at 75 ng/min (37.5 ng/min in 3 women) was reinfused for 15 minutes. Ten minutes into this infusion, the L-NMMA infusion was reinitiated at 64 µmol/min (32 µmol/min in 3 women). Measurements of coronary flow velocity and angiography were then performed before and after infusion of the same dose of acetylcholine as was used before the reinfusion of estradiol.

Statistical Analysis
Data are expressed as mean±1 SD. The acetylcholine dose that produced the greatest enhancement of coronary flow velocity during the first estradiol infusion was used for analyses of coronary dynamics during the four periods of acetylcholine stimulation of the coronary circulation in the study: (1) acetylcholine, (2) estradiol and acetylcholine, (3) L-NMMA and acetylcholine, and (4) estradiol, L-NMMA, and acetylcholine. Thus all measures of coronary responsiveness to acetylcholine before and after estradiol infusion, before and after L-NMMA administration, were performed at the same dose of acetylcholine for a given patient. Because of the duration of the study (3 hours) and the frequent need to readjust the flow wire position after prolonged infusions or rest periods to obtain an optimum flow velocity waveform, baseline measurements were performed before each of the four acetylcholine coronary stimulation studies. After testing of data for normality, each measure of coronary reactivity was analyzed over the four periods with a global ANOVA with a repeated-measures factorial design with these factors: subject, estradiol (yes, no), L-NMMA (yes, no), and an estradiol–L-NMMA interaction. In the case that the interaction term was found to be significant (that is, the estradiol effect differed before and after L-NMMA infusion), Student's paired t tests were used to compare separately the peak effect of estradiol on acetylcholine-stimulated vascular responses before and after L-NMMA infusion to block NO synthesis. All probability values are two-tailed, and a value of P<.05 is considered statistically significant.

	Results

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Intracoronary infusion of estradiol increased coronary sinus estradiol levels from 81±37 to 1725±705 pmol/L (22±10 to 470±192 pg/mL). After a 30-minute rest period, repeat intracoronary estradiol infusion increased coronary sinus estradiol levels from 114±37 to 1920±602 pmol/L (31±10 to 523±164 pg/mL), representing significant increases in resting estradiol levels (P<.001), although intrainfusion estradiol levels were similar for the two infusions (P=.22).

Coronary Vascular Effects of Estradiol
In the first 5 women enrolled in this study, the epicardial diameter, coronary flow, and coronary resistance responses to acetylcholine (30 µg/min, 3 women; 100 µg/min, 2 women) during the first intracoronary infusion of estradiol were unchanged during the second intracoronary infusion of estradiol that followed a 30-minute rest period (Table).

View this table: [in this window] [in a new window]   Table 1. Coronary Vascular Effects of Serial Intracoronary Infusions of 17ß-Estradiol in Five Postmenopausal Women

In the remaining 20 women, acetylcholine (3 µg/min, 1 woman; 30 µg/min, 8 women; 100 µg/min, 9 women; 300 µg/min, 2 women) increased coronary flow by 54±48% (from 92±67 to 139±118 mL/min), decreased epicardial luminal diameter by 8±11% (from 2.7±0.7 to 2.4±0.8 mm), and decreased coronary resistance by 32±21% (from 1.8±1.0 to 1.2±0.7 mm Hg/mL per minute), with baseline values obtained during intracoronary infusion of 5% dextrose in water. After intracoronary infusion of estradiol, acetylcholine increased coronary flow by 100±63% (from 75±57 to 154±141 mL/min) and decreased coronary resistance by 46±15% (from 2.1±1.1 to 1.1±0.7 mm Hg/mL per minute), with baseline values obtained after 15 minutes of estradiol infusion. These vasodilator responses to acetylcholine after estradiol infusion were significantly greater than the responses to the same dose of acetylcholine before estradiol infusion (both P=.007), with 14 of 20 women having a greater coronary flow response to acetylcholine during estradiol infusion than to acetylcholine alone (Fig 1). During estradiol infusion there was a trend (P=.123) toward less of an epicardial coronary artery constrictor response to acetylcholine (-3±11%, from 2.6±0.6 to 2.5±0.7 mm) compared with the response to the same dose of acetylcholine alone.

View larger version (15K): [in this window] [in a new window]   Figure 1. Coronary flow responses to acetylcholine (ACH, 100 µg/min) are plotted relative to flow responses to the same dose of ACH after infusion of estradiol (E2, 75 ng/min). Dotted line indicates the line of identity.

Estradiol Effects After Inhibition of NO Synthesis
After intracoronary infusion of L-NMMA for 5 minutes, there were insignificant changes in coronary flow (from 73±48 to 75±49 mL/min, P=.28), epicardial diameter (from 2.6±0.6 to 2.5±0.6 mm, P=.20), and coronary resistance (from 2.1±1.2 to 2.1±1.1 mm Hg/mL per minute, P=.94). During continued infusion of L-NMMA, acetylcholine increased coronary flow by 42±40% (from 75±49 to 114±99 mL/min), decreased epicardial diameter by 8±8% (from 2.5±0.6 to 2.3±0.7 mm), and decreased coronary resistance by 23±21% (from 2.1±1.1 to 1.7±1.2 mm Hg/mL per minute), with baseline values obtained after 5 minutes of L-NMMA infusion. These coronary flow and resistance responses were less than those obtained at the same dose of acetylcholine alone, although the differences did not achieve statistical significance (coronary flow, +42±48% versus +54±48%, P=.28; coronary resistance, -23±21% versus -32±21%, P=.13).

During reinfusion of estradiol for 15 minutes and L-NMMA for the last 5 minutes of this infusion, followed by acquisition of baseline values, acetylcholine increased coronary flow by 39±46% (from 70±41 to 101±71 mL/min) and decreased coronary resistance by 19±30% (from 2.2±0.9 to 1.9±1.3 mm Hg/mL per minute). The results of the global ANOVA described in the "Statistical Analysis" section showed that the coronary vascular effects of estradiol differed before and after L-NMMA for coronary blood flow and coronary vascular resistance (interaction probability values are .006 and .02, respectively) and showed a trend toward significance for coronary diameter (P=.10). The coronary responses to the combined infusions of estradiol, L-NMMA, and acetylcholine were significantly reduced compared with the responses to the same dose of acetylcholine during the initial infusion of estradiol before L-NMMA (Fig 2). Seventeen of 20 women had less of an increase in coronary flow in response to acetylcholine and estradiol during L-NMMA infusion than to acetylcholine and estradiol before L-NMMA (Fig 3). Combined administration of estradiol, L-NMMA, and acetylcholine decreased epicardial coronary artery diameter by 8±11% (from 2.6±0.6 to 2.4±0.6 mm). This magnitude of constriction was no different from the response to L-NMMA and acetylcholine before reinfusion of estradiol (P=.76) but represented greater constriction than observed during coadministration of acetylcholine and estradiol before L-NMMA, albeit of marginal statistical significance (Fig 2).

View larger version (19K): [in this window] [in a new window]   Figure 2. Epicardial coronary diameter (top), coronary blood flow (middle), and resistance (bottom) responses to acetylcholine are shown as relative changes from respective preacetylcholine baseline values in 20 postmenopausal women. The dose of acetylcholine represented on these graphs (100 µg/min) produced the coronary flow response that was maximally enhanced during coadministration of 17ß-estradiol. The effects of 17ß-estradiol (75 ng/min) on acetylcholine-stimulated changes in coronary diameter, flow, and resistance are shown before () and during () concomitant infusion of NG-monomethyl-l-arginine (L-NMMA, 64 µmol/min), an inhibitor of nitric oxide synthesis. Data are expressed as mean±SEM. Probability values are for the significance of L-NMMA effects on coronary dynamics before and during estradiol infusion.

View larger version (16K): [in this window] [in a new window]   Figure 3. Coronary flow responses to acetylcholine (ACH, 100 µg/min) during estradiol (E2, 75 ng/min) infusion are plotted relative to flow responses to the same dose of ACH after infusion of E2 and NG-monomethyl-l-arginine (L-NMMA, 64 µmol/min). Dotted line indicates the line of identity.

	Discussion

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Over the past 15 years, tremendous interest has been generated by Furchgott's observation that the endothelium importantly regulates the vasomotor tone of the underlying smooth muscle.²⁵ Subsequent studies have shown the release of vasodilating factors such as NO from the endothelium by both receptor-mediated (eg, acetylcholine, serotonin, thrombin, bradykinin) and receptor-independent (eg, shear stress) mechanisms.^{26 27 28} Considerable evidence indicates that NO activity in the systemic and coronary circulations is impaired in conditions predisposing to atherosclerosis such as hypercholesterolemia, hypertension, smoking, and aging.^{29 30 31 32 33} However, there may be sex-related differences in the impact of these conditions on endothelial function. Thus hypercholesterolemic men were found to have significantly greater impairment in acetylcholine-stimulated forearm flow compared with reproductive-aged women despite comparable elevation in LDL cholesterol levels.³⁴ Further, the age-associated decline in flow-mediated brachial artery dilation during reactive hyperemia, which stimulates the endothelium to release NO by increased shear stress, was reported to be delayed by a decade in women relative to men.³⁵ These findings suggest that sex hormones may protect endothelial function in women from conditions that impair endothelial function in men.

Consistent with our prior results in a different group of postmenopausal women,¹⁰ we found that intracoronary administration of estradiol to postmenopausal women at a dose achieving slightly supraphysiological levels of this hormone in the heart improved the epicardial and microvascular responses of the coronary circulation to the endothelium-dependent vasodilator acetylcholine in the majority of women studied. The vascular effects of estradiol are mediated through potentiation of an endothelium-dependent vasodilating mechanism, as the dose used in our study does not potentiate endothelium-independent vascular responsiveness in the coronary circulation.¹⁰

We have previously shown that L-NMMA, an arginine analogue that stereospecifically inhibits the synthesis of NO from L-arginine by NO synthase (NOS), at the intracoronary dose administered in the present study, significantly increased basal coronary resistance and attenuated the vasodilator response to acetylcholine in patients with normal coronary angiograms and no risk factors for atherosclerosis.³⁶ In contrast, 23 patients (10 women) with risk factors for atherosclerosis showed little or no response to L-NMMA in the basal state and less of a response to acetylcholine compared with the 9 patients (7 women) who had no risk factors, suggesting impaired NO synthesis or release in the coronary circulation in early atherosclerosis. The minimal effect of L-NMMA on basal and acetylcholine-stimulated coronary dynamics in our study probably is a consequence of the relatively large percentage of women who had atherosclerosis or risk factors for atherosclerosis and is compatible with impaired NO bioactivity. However, depressed endothelium-dependent responsiveness to acetylcholine does not preclude the possibility of acute improvement in this response after therapeutic intervention: Several studies have shown that L-arginine, the substrate for nitric oxide synthesis, acutely improves endothelial responses to acetylcholine.^{37 38 39}

The major finding of our study was that L-NMMA inhibited the vasodilating effect of estradiol administration on acetylcholine-stimulated epicardial and microvascular coronary vascular responses, compatible with augmentation by estradiol of the synthesis and release of NO. Alternatively, antioxidant effects of estradiol could protect NO from degradation by superoxide anions or other free radical molecules,^{40 41 42 43} resulting in increased bioactivity of NO.⁴⁴ The effect of L-NMMA on estradiol-stimulated dilator responses to acetylcholine probably is not a consequence of diminished vascular responsiveness to serial infusions of estradiol and acetylcholine over time because the first 5 women in the study showed identical responsiveness to these agonists during sequential infusions of the same duration as was used in the subsequent 20 women. Because the systemic circulation responds to acute⁹ and chronic⁴⁵ administration of estradiol similarly to the coronary circulation,¹⁰ our study provides a mechanism for the recent observation that postmenopausal women receiving estrogen therapy have higher plasma levels of nitrite/nitrate, derived in part from oxidation of NO released into the vessel lumen, than do untreated postmenopausal women.⁴⁶

Our observations support the findings of animal studies focusing on the role of NO in the vasomotor effects of estrogen. Thus aortic rings from female rats constricted to a greater degree after application of L-NMMA than aortic rings from male rats, suggesting that the aorta in female rats secretes more basal NO than male rats.¹⁴ Estradiol treatment for 5 days increased constitutive NOS activity in hearts from female guinea pigs; male animals required 5 additional days of estradiol treatment for a comparable effect.¹⁵ There was no effect of progesterone or testosterone on NOS activity in this study.

The role of the endothelium in mediating these vascular responses to estradiol is supported by recent work in several centers. Two groups have reported increases in NOS activity in endothelial cell cultures after incubation with physiological concentrations of estradiol.^{47 48} In both studies, evidence for increased NOS protein was shown by Western blotting and is compatible with estrogen responsiveness of the NOS gene promotor region at multiple half-palindromic motifs that may function as estrogen response elements.⁴⁹ Hayashi et al⁴⁸ found that the stimulatory effect of estradiol on NOS was inhibited by estrogen receptor antagonists tamoxifen and ICI 182,780. Two preliminary studies also indicate that estradiol increases constitutive NOS mRNA levels and augments NO release in human umbilical vein endothelial cells⁵⁰ and porcine and bovine aortic endothelial cells in culture after 24 hours.⁵¹ Of interest, even a 30-minute exposure to estradiol increased NO release from the endothelial cells in one of these studies.⁵⁰ In contrast to these findings, another study reported that ethinylestradiol enhanced NO bioactivity in cultured bovine aortic endothelial cells in the absence of enhanced NOS mRNA levels or activity by decreasing superoxide anion production.⁵²

Two recent studies have shown the presence of the estrogen receptor in bovine aortic and human coronary endothelial cells,^{53 54} indicating the possibility that estrogen-mediated enhancement of NOS expression or activity could be a receptor-mediated process. However, the endothelium-dependent vasomotor effects of infused estradiol are too rapid to be mediated by genomic effects of the hormone. In this regard, steroid hormones may rapidly initiate intracellular events in the absence of genomic effects, possibly by activation of receptors on the cell membrane.^{55 56 57 58} Increased intracellular calcium, as has been demonstrated in chicken and pig ovarian granulosa cells after estrogen exposure,⁵⁸ could activate NOS in endothelial cells, with enhanced synthesis and release of NO.

The results of our study differ from the report of Sudhir et al,⁵⁹ in which intracoronary estradiol dilated epicardial arteries and increased coronary flow in anesthetized dogs. This vasodilator effect of estradiol, infused at concentrations of 0.1 and 1.0 µmol/L, was not inhibited by pretreatment with N-nitro-L-arginine methyl ester to block NO synthesis or with the estrogen receptor antagonist ICI 182,780. Jiang et al⁶⁰ had previously shown that such supraphysiological doses of estrogen exert endothelium-independent dilator effects in rings from rabbit coronary arteries, probably mediated by a calcium channel inhibitory mechanism. Collins et al⁶¹ reported that coronary artery rings from oophorectomized rabbits whose estrogen therapy had been withdrawn for 48 hours relaxed to concentrations of estradiol ranging from 1 to 50 µmol/L. This vasorelaxant effect of estradiol was abolished by removal of the endothelium or inhibition of NOS. However, these concentrations of estradiol had minimal vasorelaxant effects on coronary artery rings from oophorectomized rabbits not chronically treated with estradiol. In our study, physiological concentrations of estradiol (2 nmol/L) were achieved, which have no direct vasodilating effect in the coronary circulation of chronically estrogen-deficient women.¹⁰ Estradiol at this concentration facilitates vasodilation in response to the endothelium-dependent agonist acetylcholine, without enhancement of endothelium-independent vasodilator responses to nitroprusside or adenosine.¹⁰

Increased NO bioactivity as a result of estrogen administration may not only promote smooth muscle relaxation by means of increased cGMP but also improve other important homeostatic properties of endothelium such as inhibition of activation of proinflammatory genes. In this regard, NO has been found to inhibit the activation of an important proinflammatory transcription factor, nuclear factor (NF)B.^{62 63 64} In the presence of reduced cytosolic NO or increased cytosolic oxidant stress, NFB is activated by dissociation from its inhibitor subunit (IB) after IB phosphorylation in the cytosol. It then translocates to the nucleus, where it combines with the promotor regions of several proinflammatory genes, with synthesis of gene products including cytokines, chemokines, and cell adhesion molecules. Inflammatory cells, once activated and attracted into the vessel wall by these gene products, have a variety of proatherogenic effects, including the release of reactive oxygen species, growth factors, prothrombotic factors, and in the case of monocytes, transformation into foam cells upon unregulated uptake of oxidized LDL.^{65 66} Accordingly, estrogen-mediated NO release from the vasculature may account in large part for the cardioprotective effect of estrogen, as suggested by experimental and observational studies, the proof of which awaits the completion of randomized clinical trials.

	Acknowledgments

 
We thank William Schenke, Greg Johnson, and Gloria Zalos for their technical assistance and Toni Julia for typing the manuscript.

Received February 10, 1997; revision received June 4, 1997; accepted June 8, 1997.

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