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(Circulation. 1995;91:2619-2626.)
© 1995 American Heart Association, Inc.

Articles

17ß-Estradiol Inhibits Ca²⁺ Influx and Ca²⁺ Release Induced by Thromboxane A₂ in Porcine Coronary Artery

Presented in part at the 15th Scientific Meeting of the International Society of Hypertension, Melbourne, Australia, March 22, 1994.

Shu-Zhong Han, MD, PhD; Hideaki Karaki, DVM, PhD; Yasuyoshi Ouchi, MD, PhD; Masahira Akishita, MD; Hajime Orimo, MD, PhD

From the Department of Geriatrics, Faculty of Medicine, and Department of Veterinary Pharmacology, Faculty of Agriculture (H.K.), University of Tokyo, Japan.

Correspondence to Shu-Zhong Han, MD, PhD, Department of Geriatrics, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.

	Abstract

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Background We wished to investigate the possible mechanism of the protective effect of estrogen replacement on coronary atherosclerosis observed in postmenopausal women.

Methods and Results Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and contraction were measured simultaneously in fura 2–loaded porcine coronary arterial strips stimulated by the thromboxane A₂ analogue U46619 and high-K⁺ depolarization in the presence and absence of 17ß-estradiol. Pretreatment with 17ß-estradiol (30 nmol/L to 30 µmol/L) inhibited the sustained elevation of [Ca²⁺]_i and the sustained contraction induced by 300 nmol/L U46619. Higher concentrations of 17ß-estradiol (1 to 100 µmol/L) also inhibited the U46619-induced transient increase in [Ca²⁺]_i and contraction in the absence of extracellular Ca²⁺. In the strips precontracted by 90 mmol/L K⁺, 17ß-estradiol (30 µmol/L) inhibited the increases in [Ca²⁺]_i and contraction to resting levels. In contrast, 30 µmol/L 17ß-estradiol only partially inhibited the U46619-induced sustained contraction, despite complete inhibition of the sustained increase in [Ca²⁺]_i. Verapamil (10 µmol/L) also strongly inhibited the sustained increase in [Ca²⁺]_i induced by 300 nmol/L U46619, with a partial inhibition of the U46619-induced sustained contraction. A subsequent addition of 30 µmol/L 17ß-estradiol did not show an additional inhibitory effect on either the [Ca²⁺]_i or the tension after the addition of verapamil. 17ß-Estradiol (10 µmol/L) also inhibited the increase in [Ca²⁺]_i and the contraction induced by cumulative addition of Ca²⁺ in the strips pretreated with 90 mmol/L K⁺. However, 17ß-estradiol did not change the slope of the [Ca²⁺]_i-tension curves. 17ß-Estradiol (10 µmol/L) had no effect on the levels of cAMP and cGMP in the coronary strips.

Conclusions 17ß-Estradiol inhibits the contraction of coronary vascular smooth muscle mainly by inhibiting Ca²⁺ influx without changing Ca²⁺ sensitivity of contractile elements. The Ca²⁺ channel blocker–like action of 17ß-estradiol may explain at least a part of the antiatherosclerotic effect of estrogen.

Key Words: hormones • calcium • thromboxane • arteries

	Introduction

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The remarkably low prevalence of coronary disease in premenopausal women and the loss of protection after menopause have long been noted. Estrogen replacement therapy reduces the risk of coronary atherosclerosis in postmenopausal women.^{1 2} Estrogen administration also inhibits the development and progression of experimentally induced coronary atherosclerosis in animals.^{3 4} However, the mechanism of the antiatherosclerotic effect of estrogen remains unclear. Over the past years, much attention has been focused on the possible role of estrogen on plasma lipoprotein, because estrogen has been found to lower LDL cholesterol and to raise HDL cholesterol.⁵ The lack of correlation between the lipid changes and their effect on atherosclerosis suggests that another mechanism may contribute to the cardiovascular protective effect of estrogen.^{6 7} Recent studies have made it clear that estrogen acts on the coronary vascular bed as a vasodilator substance. 17ß-Estradiol has been shown to produce coronary vasodilation in isolated perfused rabbit hearts,⁸ to cause endothelium-independent relaxation in isolated rabbit coronary arteries,^{9 10} and to restore impaired endothelium-mediated dilation of atherosclerotic coronary arteries in monkeys.³ Inhibition of vascular tone by 17ß-estradiol was previously reported to hyperpolarize smooth muscle cells,¹¹ to increase the cAMP level in smooth muscle,¹² to stimulate prostacyclin biosynthetic activity of vascular smooth muscle cells,¹³ and to be mediated by an endothelium-dependent mechanism.^{14 15} However, the exact mechanism by which estrogen modulates coronary vascular tone is not fully understood. Recent studies that measured tension development showed that 17ß-estradiol inhibits the contraction stimulated by prostaglandin F₂ and endothelin-1 in rabbit coronary arteries, possibly by inhibiting Ca²⁺ influx of smooth muscle cells.^{9 10} Since smooth muscle contraction is regulated not only by the changes in intracellular free Ca²⁺ levels ([Ca²⁺]_i) but also by the Ca²⁺ sensitivity of contractile elements,¹⁶ the simultaneous measurement of [Ca²⁺]_i and tension during contraction or relaxation is necessary to elucidate the mechanism of estrogen actions. Thus, the present study was designed to investigate the effect of 17ß-estradiol on [Ca²⁺]_i and contraction of smooth muscle cells in isolated porcine coronary arteries by simultaneous measurement of [Ca²⁺]_i and tension development.

	Methods

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Tissue Preparation and Solutions
Hearts from adult pigs of either sex were obtained from a local slaughterhouse immediately after the animals had been killed. These hearts were placed in ice-cold physiological salt solution (PSS) and brought to our laboratory. Left circumflex coronary arteries were isolated and dissected free of fat and connective tissues. The vessels were cut into spiral strips (5 mm long and 1.5 mm wide). The endothelium was removed by gentle rubbing of the intimal surface with a finger moistened with PSS. The composition of normal PSS was as follows (mmol/L): NaCl 136.9, KCl 5.4, glucose 5.5, NaHCO₃ 23.8, CaCl₂ 1.5, MgCl₂ 1.2, and EDTA 0.01. The high-K⁺ (90 mmol/L) solution was made by replacing NaCl in the normal solution by equimolar KCl. The Ca²⁺-free solution was made by omitting CaCl₂ and adding 2 mmol/L EGTA. These solutions were saturated with a mixture of 95% O₂/5% CO₂ at 37°C (pH 7.4).

Measurement of Intracellular Free Ca²⁺ Level in Smooth Muscle Cells
[Ca²⁺]_i of smooth muscle cells was measured simultaneously with muscle tension as previously described,¹⁷ with a fluorescent Ca²⁺ indicator, fura 2. Briefly, the muscle strips were loaded with 10 µmol/L acetoxymethyl ester of fura 2 (fura 2-AM) for 4 to 5 hours at 37°C. The noncytotoxic detergent Cremophor EL (0.02%) was added to increase the solubility of fura 2-AM. The concentration of Cremophor EL did not alter contraction. The fura 2–loaded strips were washed with normal PSS for 30 minutes in a tissue bath at 37°C to remove unhydrolyzed fura 2-AM. Experiments were performed with a fluorometer (CAF-100, JASCO). The muscle strip was held horizontally on a silicon rubber sheet in an organ bath containing 5 mL PSS at 37°C and bubbled with 95% O₂/5% CO₂. One end of the strip was pinned to the silicon rubber sheet, and the other end was connected to a strain-gauge transducer (Orientec) to monitor the isometric tension. The resting tension was adjusted to 1 g. The muscle strip was illuminated alternately (48 Hz) with two excitation wavelengths (340 and 380 nm) through a narrow slit in the sheet. The fluorescence emitted from the coronary strip was collected to a photomultiplier through a 500-nm filter. The intensity of the 500-nm fluorescence induced by the 340-nm excitation (F340) and that induced by the 380-nm excitation (F380) were measured, and the ratio of these two fluorescence intensities (F340/F380) was calculated. Since the dissociation constant of fura 2 for Ca²⁺ may be different from that obtained in vitro,¹⁶ we did not calculate the absolute amounts of [Ca²⁺]_i. In the present study, we thus used the relative F340/F380 ratio as an indicator of [Ca²⁺]_i, taking the ratio in resting muscle as 0% and that in 90 mmol/L K⁺–stimulated muscle as 100%. In some experiments, the agonist-stimulated sustained increase in [Ca²⁺]_i and tension in the absence of 17ß-estradiol was taken as 100%.

Measurement of cAMP and cGMP Content
The cAMP and cGMP contents of coronary smooth muscle were measured as described previously.¹⁸ Subsequent to incubation with 17ß-estradiol, the coronary strips without endothelium were frozen in liquid nitrogen and homogenized in 6% trichloroacetic acid solution. After centrifugation twice at 300 rpm, trichloroacetic acid in the supernatant was removed by washing with water-saturated ether, and the succinylated cAMP or cGMP was assayed by a competitive radioimmunoassay (Yamasa Shoyu).

Drugs
The chemicals used in this study were 17ß-estradiol (Sigma Chemical Co), U46619 (9,11-dideoxy-9,11-epoxymethanoprostaglandin F₂; Sigma), testosterone (Sigma), tamoxifen (Sigma), fura 2-AM (Dojindo Laboratories), EDTA (Dojindo), Cremophor EL (Nacalai Tesgue), indomethacin (Hoechst Japan), and N^G-monomethyl-L-arginine monoacetate (L-NMMA; Calbiochem).

Statistical Analysis
Data were analyzed by one-factor ANOVA. When statistically significant effects were found, a Newman-Keuls test was done to isolate the differences between the groups. Student's t test for unpaired data was used when appropriate. A value of P<.05 was considered significant. All data are presented in the figures as mean±SEM.

	Results

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Effects of 17ß-Estradiol on [Ca²⁺]_i and Contraction Induced by U46619 in Normal PSS
In normal PSS (containing 1.5 mmol/L Ca²⁺) (Fig 1a), 300 nmol/L U46619 induced an initial transient increase in [Ca²⁺]_i (70% of 90 mmol/L K⁺–induced sustained increase in [Ca²⁺]_i) within 30 seconds and then a sustained increase in [Ca²⁺]_i (50% of 90 mmol/L K⁺-induced sustained increase in [Ca²⁺]_i) 15 minutes after the application of U46619 in the porcine coronary arteries without endothelium. Muscle tension also reached a peak level (108% of 90 mmol/L K⁺-induced sustained contraction) within 5 minutes and then remained at a sustained level (98% of 90 mmol/L K⁺–induced sustained contraction) 15 minutes after the addition of U46619.

View larger version (20K): [in this window] [in a new window]   Figure 1. Graphs showing effects of pretreatment with various concentrations of 17ß-estradiol on the [Ca2+]i and tension development stimulated by 300 nmol/L U46619 in normal physiological salt solution in porcine coronary arteries. a, Typical recording of the changes in [Ca2+]i and tension development stimulated by 300 nmol/L U46619. W indicates wash. b, Effect of 17ß-estradiol on the [Ca2+]i (, in the absence of 17ß-estradiol, control; , 30 nmol/L; , 300 nmol/L; , 3 µmol/L; , 30 µmol/L). 17ß-Estradiol decreased the resting [Ca2+]i to the levels indicated at 0 minutes. Ordinate shows [Ca2+]i expressed as percent of 90 mmol/L K+–induced increase in [Ca2+]i. Abscissa shows time after the addition of 300 nmol/L U46619. c, Effect of 17ß-estradiol on the contraction. Symbols are same as in b. Ordinate shows tension expressed as percent of 90 mmol/L K+–induced contraction. Abscissa shows time after the addition of 300 nmol/L U46619. Each point represents mean±SEM of eight different measurements. 17ß-Estradiol was applied 5 minutes before the stimulation with 300 nmol/L U46619.

The effects of pretreatment with 17ß-estradiol on the increase in [Ca²⁺]_i and tension stimulated by 300 nmol/L U46619 are summarized in Fig 1b and 1c. 17ß-Estradiol was applied 5 minutes before stimulation with 300 nmol/L U46619. 17ß-Estradiol at a concentration >300 nmol/L significantly decreased the resting [Ca²⁺]_i (by 8% to 10% of 90 mmol/L K⁺–induced response; P<.05, n=8, Fig 1b) without changing the resting tension. 17ß-Estradiol (30 nmol/L to 30 µmol/L) significantly inhibited the sustained increase in [Ca²⁺]_i (Fig 1b) and the sustained contraction (Fig 1c) stimulated by 300 nmol/L U46619, both in a concentration-dependent manner. Similar experiments were performed with 90 mmol/L K⁺–stimulated coronary arteries, and the results are summarized in Fig 2.

View larger version (13K): [in this window] [in a new window]   Figure 2. Concentration-response curves of the inhibitory effects of 17ß-estradiol (E2) on the [Ca2+]i (a) and the contraction (b) stimulated by 300 nmol/L U46619 and 90 mmol/L K+ in porcine coronary arteries. indicates inhibition of the U46619-induced sustained increase in [Ca2+]i (a) and the sustained contraction (b) obtained from data at 20 minutes after the addition of 300 nmol/L U46619, which are shown in Fig 1b and 1c. indicates inhibition of the U46619-stimulated transient increase in [Ca2+]i (a) and the contraction (b) in Ca2+-free physiological salt solution (PSS) obtained from the experiments as shown in Fig 3. indicates inhibition of the 90 mmol/L K+–induced sustained increase in [Ca2+]i (a) and the sustained contraction (b). c, Concentration-response curves for the inhibitory effect of verapamil on the sustained contraction induced by 300 nmol/L U46619 () and 90 mmol/L K+ () in normal PSS in porcine coronary arteries. Each point represents mean±SEM of eight different measurements.

Concentration-response curves for the inhibitory effects of 17ß-estradiol on the sustained increase in [Ca²⁺]_i and the sustained contraction are shown in Fig 2a and 2b, respectively. Fig 2a shows that 17ß-estradiol induced a complete inhibition of the sustained increase in [Ca²⁺]_i stimulated both by 300 nmol/L U46619 and by 90 mmol/L K⁺ in a concentration-dependent manner. As shown in Fig 2b, 17ß-estradiol also caused a complete inhibition of the high K⁺–induced sustained contraction in a concentration-dependent manner. The maximal inhibition was 98% of that measured in the absence of 17ß-estradiol. However, 17ß-estradiol only partially inhibited the sustained contraction induced by 300 nmol/L U46619 (maximal inhibition, 67%).

Fig 2c shows the concentration-response curves for the inhibitory effects of verapamil on the sustained contraction induced by 300 nmol/L U46619 and 90 mmol/L K⁺. It is shown that high K⁺–induced sustained contraction was inhibited by verapamil completely and concentration-dependently (maximal inhibition, 99%), whereas U46619-induced sustained contraction was only partly inhibited by verapamil (maximal inhibition, 71%).

Effects of 17ß-Estradiol on [Ca²⁺]_i and Contraction Induced by U46619 in Ca²⁺-free PSS
Fig 3a and 3b shows a typical recording of the changes in [Ca²⁺]_i and contraction induced by 300 nmol/L U46619 in Ca²⁺-free PSS in the absence (3a) and presence (3b) of 17ß-estradiol. When the strip was exposed to Ca²⁺-free solution containing 2 mmol/L EGTA, the resting level of [Ca²⁺]_i gradually decreased to reach a steady level within 5 minutes, with no change in resting tension. 17ß-Estradiol was added 5 minutes before the application of U46619. In the Ca²⁺-free PSS containing 2 mmol/L EGTA, 300 nmol/L U46619 caused only a transient increase in [Ca²⁺]_i (about 79% of 90 mmol/L K⁺–induced sustained increase in [Ca²⁺]_i), reaching the maximum within 30 seconds, as well as a small contraction (Fig 3a). 17ß-Estradiol (30 µmol/L) significantly suppressed the transient increase in [Ca²⁺]_i and contraction (Fig 3b). Concentration-response curves for the inhibitory effects of 17ß-estradiol on the transient increase in [Ca²⁺]_i and contraction in the absence of external Ca²⁺ are shown in Fig 2a and 2b. The concentration of 17ß-estradiol needed to inhibit the U46619-stimulated transient increase in [Ca²⁺]_i in the absence of external Ca²⁺ was more than 10 times higher than that needed to inhibit the sustained increase in [Ca²⁺]_i in the presence of external Ca²⁺. The effect of 17ß-estradiol in the absence of external Ca²⁺ was weaker than that in the presence of external Ca²⁺.

View larger version (18K): [in this window] [in a new window]   Figure 3. Graphs showing changes in the [Ca2+]i and tension stimulated by 300 nmol/L U46619 in Ca2+-free physiological salt solution (PSS) in the absence (a) and presence (b) of 17ß-estradiol (E2) in porcine coronary arteries. 17ß-Estradiol was applied 5 minutes before the stimulation with U46619. The results are summarized in Fig 2a and 2b (). W indicates wash.

Caffeine (20 mmol/L) also induced a transient increase in [Ca²⁺]_i and a transient contraction in Ca²⁺-free PSS (90±4% of 90 mmol/L K⁺–stimulated [Ca²⁺]_i and 22±3% of 90 mmol/L K⁺–stimulated contraction, respectively; n=8). 17ß-Estradiol (1 to 100 µmol/L) had no effect on the caffeine-induced transient increase in [Ca²⁺]_i and the transient contraction (n=8).

Effects of 17ß-Estradiol on Coronary Arteries Precontracted With High K⁺ and U46619
As shown in Fig 4, high-K⁺ depolarization and 300 nmol/L U46619 caused a sustained increase in [Ca²⁺]_i and a sustained contraction. The sustained increase in [Ca²⁺]_i stimulated by U46619 was smaller than that induced by 90 mmol/L K⁺ (50% of 90 mmol/L K⁺–induced sustained increase in [Ca²⁺]_i), despite the similar magnitudes of the sustained contraction (98% of 90 mmol/L K⁺–induced sustained contraction). Addition of 17ß-estradiol (30 µmol/L) during 90 mmol/L K⁺–induced sustained contraction decreased both the [Ca²⁺]_i and the tension to resting levels (Fig 4a: for [Ca²⁺]_i, by 97.2±2.6% of control; for tension, by 95.5±3.2% of control; n=7). The [Ca²⁺]_i decreased within 2 minutes after the application of 17ß-estradiol, and the tension decreased with a slower time course. 17ß-Estradiol (30 µmol/L) also almost completely inhibited the 300 nmol/L U46619–stimulated sustained increase in [Ca²⁺]_i (by 93.4±2.9% of control; n=7), but it only partially inhibited the sustained contraction (by 58.7±2.5% of control; n=7) (Fig 4b). A subsequent addition of 10 µmol/L verapamil did not show any additional inhibitory effect on [Ca²⁺]_i and contraction. Fig 4c shows the effect of 10 µmol/L verapamil applied during the sustained contraction induced by 300 nmol/L U46619. Verapamil (10 µmol/L) strongly inhibited the U46619-stimulated sustained increase in [Ca²⁺]_i (by 96.1±3.6% of control; n=7), with a partial inhibition of the sustained contraction (by 54.6±3.3% of control; n=7). A subsequent addition of 30 µmol/L 17ß-estradiol did not show an additional inhibitory effect on either [Ca²⁺]_i or contraction.

View larger version (17K): [in this window] [in a new window]   Figure 4. Graphs showing effects of 17ß-estradiol (E2) and verapamil on the coronary arteries precontracted with 90 mmol/L K+ (a) and 300 nmol/L U46619 (b, c). 17ß-Estradiol (30 µmol/L) and verapamil (10 µmol/L) were added when the increase in [Ca2+]i and tension induced by high K+ (a) or U46619 (b) reached a steady level (n=7 for each). W indicates wash.

Effects of 17ß-Estradiol on the [Ca²⁺]_i-Tension Relation
Fig 5 shows a typical recording of the changes in smooth muscle [Ca²⁺]_i and contraction induced by increasing the extracellular Ca²⁺ during high-K⁺ depolarization in the absence (Fig 5a) and presence (Fig 5b) of 17ß-estradiol. After a 5-minute incubation with Ca²⁺-free PSS containing 2 mmol/L EGTA and then incubation with Ca²⁺-free PSS without EGTA, the extracellular Ca²⁺ was increased cumulatively (0.03 to 10.0 mmol/L). 17ß-Estradiol was applied 5 minutes before the cumulative addition of Ca²⁺. Results of the experiments are summarized in Fig 6. The cumulative application of Ca²⁺ during high-K⁺ depolarization increased both [Ca²⁺]_i and tension, and there was a positive correlation between the two parameters. Pretreatment with 10 µmol/L 17ß-estradiol strongly inhibited both [Ca²⁺]_i and tension (Fig 5b), although the slope of the [Ca²⁺]_i-tension curve did not change (Fig 6).

View larger version (18K): [in this window] [in a new window]   Figure 5. Graphs showing changes in the [Ca2+]i and tension induced by the cumulative addition of Ca2+ during 90 mmol/L K+ depolarization in the absence (a) and presence (b) of 17ß-estradiol (E2; 10 µmol/L) in porcine coronary arteries. Numbers at each triangle indicate the concentrations of extracellular Ca2+ (-log M, where M is concentration in mol/L). 17ß-Estradiol was applied 5 minutes before the cumulative application of Ca2+. The results are summarized in Fig 6. W indicates wash.

View larger version (21K): [in this window] [in a new window]   Figure 6. Graph showing effects of 17ß-estradiol on [Ca2+]i-tension relation in porcine coronary arteries ( and ). Results were obtained by the cumulative addition of Ca2+ during high-K+ depolarization in the absence () and presence () of 10 µmol/L 17ß-estradiol, as shown in Fig 5 (n=8 for each). indicates results obtained by the cumulative addition of Ca2+ in the presence of 300 nmol/L U46619 (n=7). indicates results obtained by the addition of 300 nmol/L U46619 in the presence of 17ß-estradiol, as shown in Fig 1 (n=7). Concentrations of 17ß-estradiol applied were 0, 30 nmol/L, 300 nmol/L, 3 µmol/L, and 30 µmol/L, from the upper right to the lower left open squares.

Fig 6 also illustrates the [Ca²⁺]_i-tension relation obtained by the cumulative addition of Ca²⁺ in the presence of 300 nmol/L U46619. It is shown that despite a good correlation between [Ca²⁺]_i and tension in the presence of U46619, the [Ca²⁺]_i-tension relation is located to the left of that obtained in the presence of high K⁺. This indicates that a greater contraction was induced in the presence of 300 nmol/L U46619 than in the presence of high K⁺ for a given increase in [Ca²⁺]_i. Fig 6 shows the [Ca²⁺]_i-tension curve obtained by the addition of 300 nmol/L U46619 in the presence of 30 nmol/L to 30 µmol/L 17ß-estradiol, the data for which are shown in Fig 1b and 1c. The result indicates that although both [Ca²⁺]_i and contraction were inhibited, the [Ca²⁺]_i-tension relation was not changed by 17ß-estradiol.

Effect of 17ß-Estradiol on cAMP and cGMP Contents
Pretreatment of porcine coronary arterial strips with 17ß-estradiol for 60 minutes had no effect on the levels of cAMP (control, 233±14 pmol/g wet wt; 17ß-estradiol treatment, 256±16 pmol/g wet wt) or levels of cGMP (control, 15±1.8 pmol/g wet wt; 17ß-estradiol treatment, 17.5±1.5 pmol/g wet wt).

	Discussion

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Thromboxane A₂ (TXA₂) is known to be a major cyclooxygenase-dependent endothelium-derived contracting factor and a potent constrictor of coronary smooth muscle as well as a strong inducer of platelet aggregation. TXA₂ was reported to cause coronary contraction in vitro and coronary vasospasm in vivo and is though to be involved in coronary atherosclerosis.^{19 20 21} U46619 is a full TXA₂ receptor agonist.²²

In the presence of extracellular Ca²⁺, U46619 induced a transient increase followed by a sustained increase in [Ca²⁺]_i and a sustained contraction in the isolated porcine coronary arteries (Fig 1). In the absence of extracellular Ca²⁺, U46619 caused only a transient elevation of [Ca²⁺]_i and a small, sustained contraction (Fig 3). Because the sustained increase in [Ca²⁺]_i but not the initial transient increase in [Ca²⁺]_i was completely inhibited by the removal of external Ca²⁺, it is generally accepted that the transient increase in [Ca²⁺]_i is due to the Ca²⁺ release from intracellular stores, whereas the sustained increase in [Ca²⁺]_i is due to the increase in transmembrane Ca²⁺ influx. Since the sustained increase in [Ca²⁺]_i was almost completely inhibited by the Ca²⁺ channel blocker verapamil (Fig 4c), U46619 seems to open voltage-dependent Ca²⁺ channels (VDCs) to increase Ca²⁺ influx. This result is consistent with the observations that receptor agonists open VDCs to induce a sustained increase in [Ca²⁺]_i.^{17 23 24 25} Pretreatment with 17ß-estradiol concentration-dependently and completely inhibited the U46619-stimulated sustained increase in [Ca²⁺]_i, indicating that 17ß-estradiol inhibits U46619-stimulated Ca²⁺ influx through VDCs.

High-K⁺ depolarization has been shown to increase [Ca²⁺]_i by activating VDCs, which are inhibited by Ca²⁺ channel blockers. 17ß-Estradiol (30 µmol/L) completely inhibited the high-K⁺–induced increase in [Ca²⁺]_i and contraction to resting levels (Figs 2 and 4a). This result indicates that 17ß-estradiol inhibits the Ca²⁺ influx through VDCs to inhibit the high-K⁺–induced contraction. Although 17ß-estradiol strongly inhibited the Ca²⁺ influx stimulated by 300 nmol/L U46619, it only partially inhibited contraction (Fig 4b). A portion of the U46619-induced contraction not inhibited by 17ß-estradiol was also insensitive to verapamil (Fig 4b), whereas a portion of the U46619-induced contraction not inhibited by verapamil was also insensitive to 17ß-estradiol (Fig 4c). These findings suggest that 17ß-estradiol and verapamil are acting on the same mechanism. To examine the reason why 17ß-estradiol only partially inhibited the contraction induced by U46619, we constructed the [Ca²⁺]_i-tension curves. The results show that the [Ca²⁺]_i-tension relation obtained in the presence of 300 nmol/L U46619 is located to the left of that obtained in the presence of high K⁺ (Fig 6). This observation that 300 nmol/L U46619 induces a greater contraction than 90 mmol/L K⁺ for a given increase in [Ca²⁺]_i indicates that 300 nmol/L increases the Ca²⁺ sensitivity of contractile elements. A similar increase in Ca²⁺ sensitivity has been reported with other receptor agonists. It has been demonstrated that receptor agonists increase not only the [Ca²⁺]_i by opening Ca²⁺ channels and mobilizing Ca²⁺ release but also the sensitivity of myosin light chain phosphorylation to [Ca²⁺]_i through a G protein–mediated pathway, resulting in a greater contraction than that induced by high-K⁺ depolarization for a given increase in [Ca²⁺]_i.^{17 26 27} It has also been reported that vascular smooth muscle relaxation is regulated by the decrease in [Ca²⁺]_i and/or the decrease in Ca²⁺ sensitivity of contractile elements.^{18 28 29} The [Ca²⁺]_i-tension curves in Fig 6 indicate that although 17ß-estradiol inhibited the increase in [Ca²⁺]_i and the contraction induced by high K⁺ or U46619, it did not change the [Ca²⁺]_i-tension relation, suggesting that 17ß-estradiol does not change [Ca²⁺]_i sensitivity. Previous reports have shown that Ca²⁺ channel blockers, such as verapamil and nifedipine, inhibited transmembrane Ca²⁺ influx through VDCs but not Ca²⁺ sensitivity.^{25 30 31} From these results, we conclude that 17ß-estradiol has an effect similar to that of Ca²⁺ channel blockers: inhibition of VDCs.

In the present study, we also found that 17ß-estradiol suppressed the U46619-induced transient increase in [Ca²⁺]_i (Fig 3), suggesting that 17ß-estradiol inhibits U46619-induced Ca²⁺ release from intracellular stores. The concentration of 17ß-estradiol needed to inhibit the U46619-induced Ca²⁺ release was more than 10 times higher than that needed to inhibit the U46619-induced increase in Ca²⁺ influx, which suggests that 17ß-estradiol has a concentration-dependent dual mechanism of action. 17ß-Estradiol did not inhibit the caffeine-induced transient increase in [Ca²⁺]_i and contraction in Ca²⁺-free PSS even at a very high concentration (100 µmol/L). Because Ca²⁺ release induced by the agonist U46619 is attributable to receptor-mediated formation of inositol 1,4,5-triphosphate (IP₃), whereas caffeine-induced Ca²⁺ release is due to a Ca²⁺-induced Ca²⁺ release mechanism,^{32 33} high concentrations of 17ß-estradiol may selectively inhibit the Ca²⁺ release induced by IP₃ or inhibit the receptor-linked signal transduction pathway.

Increases in the levels of cyclic nucleotides such as cAMP or cGMP in vascular smooth muscle cells have been shown to inhibit receptor-mediated Ca²⁺ release. However, 17ß-estradiol did not affect the content of cAMP or cGMP. Furthermore, we found that indomethacin or L-NMMA did not change 17ß-estradiol–induced relaxation (data not shown), suggesting that the stimulation of prostacyclin synthesis or nitric oxide release is not involved in the inhibitory effect of 17ß-estradiol on coronary smooth muscle contraction.

It is not clear whether the inhibitory effect of 17ß-estradiol observed in our study is mediated by an estrogen receptor. Specific cytosolic-nuclear binding sites for 17ß-estradiol have been found in vascular smooth muscle cells, including rat aortic smooth muscle cell cultures^{34 35} and canine coronary smooth muscles.¹¹ Although the membrane estrogen receptor has not been identified in vascular smooth muscle cells, previous studies have shown that several kinds of cells of some mammal species may bear nongenomic cell surface receptor for 17ß-estradiol.^{36 37} In our study, 17ß-estradiol inhibited the increase in [Ca²⁺]_i and contraction within 5 minutes. The rapid time course of action of 17ß-estradiol is inconsistent with that mediated by conventional slow-acting nuclear estrogen receptors. Furthermore, pretreatment with 10 µmol/L tamoxifen, a potent inhibitor of genomic estrogen responses, did not change the effect of 17ß-estradiol (data not shown). Thus, 17ß-estradiol may act on a cell membrane receptor yet to be identified. It should also be pointed out that the effects presented here were produced by a short-term application of high concentrations of 17ß-estradiol to in vitro coronary smooth muscle tissues. Effects of the long-term application of physiological concentrations of estrogen in vivo need to be investigated.

It is well known that Ca²⁺ channel blockers prevent the development and progression of coronary atherosclerosis both in animals and in humans.^{38 39 40} Ca²⁺ channel blockers not only attenuate the atherosclerotic lesions but also preserve the endothelium-dependent relaxation that would be impaired in cholesterol-fed rabbits.⁴¹ Similarly, estrogen replacement therapy protects against coronary artery atherosclerosis^{1 2 4} and modulates impaired endothelium-mediated dilation of atherosclerotic coronary arteries.³ These protective effects of estrogen replacement may involve its Ca²⁺ channel blocker mechanism.

In summary, 17ß-estradiol inhibited the Ca²⁺ influx stimulated by the receptor agonist U46619 and the high-K⁺ depolarization in isolated porcine coronary arteries without changing the Ca²⁺ sensitivity of contractile elements. These effects are similar to those observed with Ca²⁺ channel blockers. 17ß-Estradiol also inhibited the Ca²⁺ release induced by agonist U46619 at higher concentration. The Ca²⁺ channel blocker–like effect of 17ß-estradiol may explain at least a part of the cardiovascular protective effect of estrogen.

Received November 7, 1994; accepted December 12, 1994.

	References

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1. Sullivan JM, Vander-Zwaag R, Lemp GF, Hughes JP, Maddock V, Kroetz FW, Ramanathan KB, Mirvis DM. Postmenopausal estrogen use and coronary atherosclerosis. Ann Intern Med. 1988;108:358-363.

2. Stampfer MJ, Golditz GA, Willett WC, Manson JE, Rosner B, Speizer FE, Hennekens CH. Postmenopausal estrogen therapy and cardiovascular disease. N Engl J Med. 1991;325:756-762. [Abstract]

3. Williams JK, Adams MR, Klopfenstein HS. Estrogen modulates responses of atherosclerotic coronary arteries. Circulation. 1990;81:1680-1687. [Abstract/Free Full Text]

4. Hough JL, Zilversmit DB. Effect of 17ß-estradiol on aortic cholesterol content and metabolism in cholesterol-fed rabbits. Atherosclerosis. 1986;6:57-63.

5. Bush TL, Cowan LD, Barrett-Connor E. Estrogen use and all-cause mortality: preliminary results from the Lipid Research Clinics Program Follow-up Study. JAMA. 1983;249:814-820.

6. Clarkson TB, Shively CA, Morgan TM, Koritnik DR, Adams MR, Kaplan JR. Oral contraceptives and coronary artery atherosclerosis of cynomolgus monkeys. Obstet Gynecol. 1990;75:217-222. [Medline] [Order article via Infotrieve]

7. Adams MR, Clarkson JR, Kaplan JR, Koritnik DR. Ovarian secretions and atherosclerosis. In: Naftoli F, Gutmann JN, Decherney AH, Sarrel PM, eds. Ovarian Secretion and Cardiovascular and Neurological Function. New York, NY: Raven Press; 1990:151-159.

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

9. Jiang C, Sarrel PM, Lindsay DC, Poole-Wilson PA, Collins P. Endothelium-independent relaxation of rabbit coronary artery by 17ß-estradiol in vitro. Br J Pharmacol. 1991;104:1033-1037. [Medline] [Order article via Infotrieve]

10. Jiang C, Sarrel PM, Poole-Wilson PA, Collins P. Acute effect of 17ß-estradiol on rabbit coronary artery contractile responses to endothelin-1. Am J Physiol. 1992;263:H271-H275. [Abstract/Free Full Text]

11. 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]

12. Kishi Y, Numano F. A study of the mechanism of estrogen as an antiatherosclerotic: the inhibitory effect of estrogen on A23187-induced contraction of the aortic wall. Mech Ageing Dev. 1981;18:115-123.

13. Chang WC, Nakao J, Orimo H, Murota S. Stimulation of prostacyclin biosynthetic activity by estradiol in rat aortic smooth muscle cells in culture. Biochim Biophys Acta. 1980;619:107-113. [Medline] [Order article via Infotrieve]

14. Gisclad V, Miller VM, Vanhoutte PM. Effect of 17ß-estradiol on endothelium-dependent responses in the rabbit. J Pharmacol Exp Ther. 1988;244:19-22. [Abstract/Free Full Text]

15. Van Buren G, Yang D, Clark KE. Estrogen-induced uterine vasodilation is antagonized by L-nitroarginine methyl ester, an inhibitor of nitric oxide synthesis. Am J Obstet Gynecol. 1992;16:828-833.

16. Karaki H. Ca⁺⁺ localization and Ca⁺⁺ sensitivity in vascular smooth muscle. Trends Pharmacol Sci. 1989;10:320-325. [Medline] [Order article via Infotrieve]

17. Karaki H, Sato K, Ozaki H. Different effects of norepinephrine and KCl on the cytosolic Ca⁺⁺-tension relationship in vascular smooth muscle of rat aorta. Eur J Pharmacol. 1988;151:325-328. [Medline] [Order article via Infotrieve]

18. Abe A, Karaki H. Inhibitory effects of forskolin on vascular smooth muscle of rabbit aorta. Jpn J Pharmacol. 1988;46:293-301. [Medline] [Order article via Infotrieve]

19. Moncada S, Vane JR. Pharmacology and endogenous roles of prostaglandin endoperoxides, thromboxane A₂ and prostacyclin. Pharmacol Rev. 1979;30:293-331. [Medline] [Order article via Infotrieve]

20. Lewy RI, Wiener L, Walinsky P, Lefer AM, Silver MJ, Smith JB. Thromboxane release during pacing-induced angina pectoris: possible vasoconstrictor influence on the coronary vasculature. Circulation. 1980;61:1165-1171. [Free Full Text]

21. Ellis EF, Oelz O, Roberts LJ, Payne NA, Sweetman BJ, Nies AS, Oates JA. Coronary arterial smooth muscle contraction by a substance released from platelets: evidence that it is thromboxane A₂. Science. 1976;193:1135-1137. [Abstract/Free Full Text]

22. Hanasaki K, Nakano K, Kasai H, Arita H, Ohtani K, Doteuchi M. Specific receptors for thromboxane A₂ in cultured vascular smooth muscle cells of rat aorta. Biochem Biophys Res Commun. 1988;150:1170-1175. [Medline] [Order article via Infotrieve]

23. Nelson MT, Standen NB, Brayden JE, Worley JF III. Noradrenaline contracts arteries by activating voltage-dependent calcium channels. Nature. 1988;336:382-385. [Medline] [Order article via Infotrieve]

24. Ohya Y, Sperelakis N. Involvement of a GTP-binding protein in stimulating action of angiotensin II on calcium channels in vascular smooth muscle cells. Circ Res. 1991;68:763-771. [Abstract/Free Full Text]

25. Karaki H, Sato K, Ozaki H. Different effects of verapamil on cytosolic Ca⁺⁺ and contraction in norepinephrine-stimulated vascular smooth muscle. Jpn J Pharmacol. 1991;55:35-42. [Medline] [Order article via Infotrieve]

26. Rembold CM, Murphy RA. Myoplasmic [Ca⁺⁺]_i determines myosin phosphorylation in agonist-stimulated swine arterial smooth muscle. Circ Res. 1988;63:593-603. [Abstract/Free Full Text]

27. Kitazawa T, Gaylinn BD, Denney GH, Somlyo AP. G-protein-mediated Ca⁺⁺ sensitization of smooth muscle contraction through myosin light-chain phosphorylation. J Biol Chem. 1991;266:1708-1715. [Abstract/Free Full Text]

28. Rembold CM. Regulation of contraction and relaxation in arterial smooth muscle. Hypertension. 1992;20:129-137. [Abstract/Free Full Text]

29. Karaki H, Sato K, Ozaki H, Murakami K. Effects of sodium nitroprusside on cytosolic calcium level in vascular smooth muscle. Eur J Pharmacol. 1988;156:259-266. [Medline] [Order article via Infotrieve]

30. Yamagishi T, Yanagisawa T, Taira N. Ca⁺⁺ influx induced by agonist U46619 is inhibited by hyperpolarization induced by the K⁺ channel opener cromakalim in canine coronary artery. Jpn J Pharmacol. 1992;59:291-299. [Medline] [Order article via Infotrieve]

31. Schachtele C, Wagner B, Rudolph C. Effect of Ca⁺⁺ entry blockers on myosin light chain kinase and protein kinase C. Eur J Pharmacol. 1989;163:151-155. [Medline] [Order article via Infotrieve]

32. Bond M, Kitazawa T, Somlyo AP, Somlyo AV. Release and recycling of calcium by the sarcoplasmic reticulum in guinea-pig portal vein smooth muscle. J Physiol (Lond). 1984;355:677-695. [Abstract/Free Full Text]

33. Karaki H, Weiss GB. Calcium release in smooth muscle. Life Sci. 1988;42:111-122. [Medline] [Order article via Infotrieve]

34. Nakao J, Chang WC, Murota SI, Orimo H. Estradiol-binding sites in rat aortic smooth muscle cells in culture. Atherosclerosis. 1981;38:75-80. [Medline] [Order article via Infotrieve]

35. Orimo A, Inoue S, Ikegami A, Hosoi T, Akisita M, Ouchi Y, Muramatsu M, Orimo H. Vascular smooth muscle cells as target for estrogen. Biochem Biophys Res Commun. 1993;195:730-736. [Medline] [Order article via Infotrieve]

36. Pietras RJ, Szego CM. Estrogen receptors in uterine plasma membrane. J Steroid Biochem. 1979;11:1471-1483. [Medline] [Order article via Infotrieve]

37. Matsuda S, Kadowaki Y, Ichino M, Akiyama T, Toyoshima K, Yamamoto T. 17ß-Estradiol mimics ligand activity of the c-er B2 protooncogene product. Proc Natl Acad Sci U S A. 1993;90:10803-10807. [Abstract/Free Full Text]

38. Henry PD, Bentley KI. Suppression of atherogenesis in cholesterol-fed rabbit treated with nifedipine. J Clin Invest. 1981;68:1366-1369.

39. Rouleau JL, Parmley WW, Stevens J, Wikman-Coffelt J, Sievers R, Mahley RW, Havel RJ, Brecht W. Verapamil suppresses atherosclerosis in cholesterol-fed rabbits. J Am Coll Cardiol. 1983;1:1453-1460. [Abstract]

40. Lichtlen PR, Hugenholtz PG, Rafflenbeul W, Hecker H, Jost S, Deckers JW. Retardation of angiographic progression of coronary artery disease by nifedipine: results of the International Nifedipine Trial on Antiatherosclerotic Therapy (INTACT). Lancet. 1990;335:1109-1113. [Medline] [Order article via Infotrieve]

41. Kappagoda CT, Thomson AB, Senaratne MP. Effect of nisoldipine on atherosclerosis in the cholesterol fed rabbit: endothelium dependent relaxation and cholesterol content. Circ Res. 1991;25:270-282.

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