Introduction

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Gender has been recognized as an important factor in determining the risk of coronary heart disease (CHD; Barret-Connor and Bush, 1991). Although CHD claims the lives of approximately 500,000 women in the United States every year, the incidence of CHD is relatively low among premenopausal women with a sharp rise after menopause (Barret-Connor and Bush, 1991; Stampfer et al., 1991). The increased risk of CHD in young women after bilateral oophorectomy and the beneficial effects of estrogen replacement therapy in postmenopausal women have suggested a role for estrogen in protecting against the development of CHD (Stampfer et al., 1991).

Estrogen may protect against cardiovascular diseases by exerting several beneficial effects such as modification of the composition of circulating lipoproteins (Kushwaha and Hazzard, 1981), changes in blood coagulation (Bing and Conforto, 1992), inhibition of intravascular accumulation of collagen (Wolinsky, 1972), antiproliferative effects on vascular smooth muscle (Clowes et al., 1983), and direct cardiovascular protective effects on the hemodynamics (Williams et al., 1992). Estrogens are vasodilators; for example, estrogen causes vasodilation in deendothelialized rabbit coronary artery precontracted by endothelin-1, prostaglandin F₂ (PGF₂), or high KCl depolarizing solution (Jiang et al., 1991), suggesting that the estrogen-induced inhibition of vascular tone has an endothelium-independent component that involves direct action on vascular smooth muscle (Harder and Coulson, 1979; Gerhard and Ganz, 1995; Farhat et al., 1996).

In contrast with the well known vasodilator effects of estrogen, the vascular effects of other sex hormones, such as progesterone and testosterone, are less clear. Also, the mechanisms involved in the vascular smooth muscle relaxation by sex hormones have not been clearly identified. The rapid vascular effects of estrogen have suggested additional mechanisms independent of the classic genomic pathway of steroid action, which involves gene transcription (Landers and Spelsberg, 1992). Vascular smooth muscle contraction has largely been explained by increases in [Ca²⁺] due to initial Ca²⁺ release from the intracellular stores and maintained Ca²⁺ entry from the extracellular space (Khalil and van Breemen, 1995). The rapid effects of 17-estradiol on vascular contractility suggest that it may be mediated by an effect on Ca²⁺ mobilization and/or fluxes.

Because of the multiplicity of the vascular effects of sex hormones on various types of vascular cells, study of the effects and mechanisms of action of sex hormones in a multicellular vascular preparation such as the coronary artery could be difficult. Therefore, the purposes of the present study were to determine whether: 1) sex hormones cause relaxation in single coronary smooth muscle cells, 2) the sex hormone-induced changes in cell contraction reflect changes in [Ca²⁺]_i, and 3) the sex hormone-induced changes in [Ca²⁺]_i, if any, are due to changes in Ca²⁺ release from the intracellular stores and/or Ca²⁺ entry from the extracellular space. To avoid the possible influence of circulating levels of sex hormones on the [Ca²⁺]_i measurements and to investigate possible gender differences in the responses, this study was performed primarily on coronary smooth muscle cells of gonadectomized male pigs in comparison with some of the measurements in cells from gonadectomized female pigs.

	Materials and Methods

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Tissue Preparation. Gonadectomized prepubertal male and female Yorkshire pigs (12 weeks old, 30 kg) were purchased from a local breeder. The pigs were gonadectomized at 8 weeks of age and studied 4 weeks later. The pigs were anesthetized by inhalation of isoflurane (Ohio Medical Products, Madison, WI), the abdominal cavity was exposed through a ventral midline incision, and the animal was bled by severing both the descending aorta and the inferior vena cava. The thoracic cavity was opened, and the heart was rapidly excised from the pericardial sac and placed in normal Krebs' solution. With the use of a dissection microscope, the left anterior descending coronary artery was dissected and cleaned of connective and adipose tissue. The coronary artery was opened by cutting along its longitudinal axis, and the endothelium was removed by gently rubbing the vessel interior with wet filter paper. The tissue was then sectioned into approximately 2 × 2-mm strips. All procedures were conducted following the guidelines of the Animal Care and Use Committee at the University of Mississippi Medical Center and the American Physiological Society.

Single Cell Isolation. Single coronary smooth muscle cells were freshly isolated as described previously, specifically avoiding aspiration through a pipette or centrifugation (Khalil and Morgan, 1992). Coronary artery strips (50 mg) were placed in a siliconized flask containing a tissue digestion mixture of collagenase type II (236 U/mg protein activity; Worthington, Freehold, NJ), elastase grade II (3.25 U/mg protein activity; Boehringer Mannheim, Indianapolis, IN), and trypsin inhibitor type II-soybean (10,000 U/ml; Sigma Chemical Co., St. Louis, MO) in 7.5 ml of Ca²⁺- and Mg²⁺-free Hanks' solution supplemented with 30% BSA (Sigma). The tissue was incubated three consecutive times in the tissue digestion mixture to yield three separate batches of cells. For the first batch, the tissue was incubated with 5 mg of collagenase, 4 mg of elastase, and 147 µl of trypsin inhibitor for 60 min. For batches 2 and 3, the collagenase was reduced to 2.5 mg, the trypsin inhibitor was reduced to 122 µl, and the incubation period was reduced to 30 min. The tissue preparation was placed in a shaking water bath at 34°C in an atmosphere of 95% O₂/5% CO₂. At the end of each incubation period, the preparation was rinsed with 12.5 ml of Hanks' solution with albumin and poured over glass coverslips placed in wells and cooled to 2°C. By using the gravitational force, the cells were allowed to settle and adhere to the glass coverslips. Ca²⁺ was gradually added back to the preparation to avoid the "calcium paradox" (Nayler et al., 1984). The cell isolation procedure produced cells of varying lengths. Only long spindle-shaped cells of 70 µm in length were selected for this study.

Contractility Studies. Four different smooth muscle activators were used in the present study. PGF₂ was used as one of the potent vasoactive eicosanoids that have been suggested to be released in response to tissue injury and have been implicated in the pathogenesis of coronary vasospasm. Caffeine and carbachol were used to activate the Ca²⁺ release mechanism in Ca²⁺-free solution (Leijeten and van Breemen, 1984; Takuwa et al., 1986). Membrane depolarization by high KCl solution was used to activate the Ca²⁺ entry mechanism from the extracellular space (Khalil and van Breemen, 1995). The changes in cell length in response to PGF₂ (10⁵ M), caffeine (10 mM), carbachol (10⁵ M), and high KCl depolarizing solution (51 mM) were measured in freshly isolated coronary smooth muscle cells untreated or pretreated with one of the sex hormones for 30 min or 1 h and viewed under the microscope using a Nikon 40× objective. The magnitude of cell shortening was expressed as the final cell length (L) as a fraction of the initial cell length (L_i). All contraction measurements were made at 22°C as described previously (Khalil and Morgan, 1992).

Measurement of [Ca²⁺]_i. [Ca²⁺]_i was measured in Fura-2-loaded single coronary smooth muscle cells using the ratio method as described previously (Williams et al., 1987; Khalil et al., 1994). The cells were incubated in the Fura-2 loading solution for 30 min at 34°C. The loading solution consisted of normal Hanks' solution supplemented with 1 µM concentration of the cell-permeant Fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR) and 0.01% Pluronic F-127 (Sigma). The Fura-2 acetoxymethyl ester was diluted from a 1 mM stock solution in dimethyl sulfoxide so that the final concentration of dimethyl sulfoxide in the loading solution was 0.1%. The Fura-2-loaded cells were washed twice and further incubated in normal Hanks' solution for at least 30 min to allow complete deesterification of the dye. Nonspecific intracellular esterases hydrolyze the AM esters and liberate the Ca²⁺-sensitive indicator (Grynkiewicz et al., 1985). Due to the photosensitivity of the Fura-2 molecule, precautionary measures were taken throughout the procedure to avoid extensive photobleaching.

(1)

2

5

2

2

5

Solutions. Krebs' solution was used for dissecting the tissue and contained 120 mM NaCl, 5.9 mM KCl, 25 mM NaHCO₃, 1.2 mM NaH₂PO₄, 11.5 mM dextrose, 2.5 mM CaCl₂, and 1.2 mM MgCl₂. The solution was bubbled for 30 min with a 95% O₂/5% CO₂ mixture to adjust the pH to 7.4. Hanks' solution was used for cell isolation and for the experiments and contained 137 mM NaCl, 5.4 mM KCl, 0.44 mM KH₂PO₄, 0.42 mM Na₂HPO₄, 4.17 mM NaHCO₃, 5.55 mM dextrose, and 10 mM HEPES. The solution was bubbled for 30 min with a 95% O₂/5% CO₂ mixture, and NaOH was added to adjust the solution pH to 7.4. For Ca²⁺- and Mg²⁺-containing Hanks' solution, 1 mM CaCl₂ and 1.2 mM MgCl₂ were added. For Ca²⁺-free Hanks' solution, CaCl₂ was omitted and replaced with 2 mM EGTA. The high KCl depolarizing solution had the same composition as normal Krebs' solution with equimolar substitution of NaCl with KCl.

Drugs and Chemicals. Stock solutions of PGF₂ (10² M, Sigma) and carbachol (10¹ M carbamylcholine chloride; Sigma) were prepared in distilled water. Caffeine (Sigma) was prepared as a 10-mM concentration in Ca²⁺-free (2 mM) EGTA Hanks' solution. Stock solution of 17-estradiol (2,3,5[10]-estratriene-3,17-diol; Sigma) was prepared as 5 × 10² M in 100% ethanol. Stock solutions of progesterone (4-pregnene-3,20-dione; Sigma) and testosterone (4-androsten-17-ol-3-one; Sigma) were prepared as 10¹ M in 100% ethanol. 17-Estradiol, tamoxifen, RU-486 (mifepristone), and flutamide were purchased from Sigma and prepared as 10² M stock solutions in 100% ethanol. ICI 182,780 (Tocris, Ballwin, MO) was prepared as 10² stock solution in 100% ethanol. The final concentration of the vehicle ethanol in solution was 0.001%. All other chemicals were of reagent grade or better.

Statistical Analysis. The data were analyzed and presented as the mean ± S.E. Results were compared using one-way ANOVA with Scheffé's F test and Student's t test for unpaired data with p < .05 considered statistically significant

	Results

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Freshly isolated coronary smooth muscle cells were responsive to contractile stimuli. In normal Hanks' (1 mM Ca²⁺) solution, PGF₂ (10⁵ M) caused contraction of coronary smooth muscle cells of gonadectomized male pigs and decreased cell length to 0.73 ± 0.02 (n = 35) of the initial length (Table 1). We examined the effects of pretreatment of the cells with 17-estradiol, progesterone, or testosterone for 30 min on cell contraction (Fig. 1A). Pretreatment of the cells with increasing concentrations of the sex hormones did not cause any change in cell length. Pretreatment of the cells with increasing concentrations of 17-estradiol caused concentration-dependent inhibition of PGF₂-induced contraction, and at a 10⁷ M concentration, 17-estradiol significantly decreased the magnitude of cell contraction in response to 0.94 ± 0.01 (n = 16) of the initial cell length (Table 1). The PGF₂-induced contraction in the presence of 10⁷ M 17-estradiol was 22.22% of maximal PGF₂ contraction in the absence of the hormone. Washing away of 17-estradiol and then the addition of fresh PGF₂ (10⁵ M) caused significant cell contraction to 0.74 ± 0.02 (n = 15) of original cell length. This contraction was not significantly different from the PGF₂ (10⁵ M) contraction in cells that were not previously exposed to 17-estradiol. Pretreatment of the cells with 17-estradiol (10⁷ M) for longer periods of time (1 h) and then washing the hormone away did not significantly affect the contractile response to PGF₂ (10⁵ M). Pretreatment of the cells with 17-estradiol (10⁷ M) for 30 min did not significantly inhibit the contractile response in the cells. The PGF₂ (10⁵ M) contraction in the presence of 17-estradiol (0.75 ± 0.02, n = 26) was not significantly different from that in the absence of the hormone. Pretreatment of the cells with the estrogen receptor antagonist tamoxifen (10⁶ M) or ICI 182,780 (10⁶ M) for 30 min completely abolished the inhibition of PGF₂ contraction by 10⁷ M 17-estradiol. The PGF₂ (10⁵ M) contraction in the presence of 17-estradiol plus tamoxifen (0.73 ± 0.02, n = 19) or in the presence of 17-estradiol plus ICI 182,780 (0.75 ± 0.03, n = 10) was not significantly different from that in the absence of the hormone and the antagonist. In the absence of sex hormones, the PGF₂ (10⁵ M)-induced contraction in cells of gonadectomized female pigs was not significantly different from that in cells of gonadectomized males (Table 1). In addition, the inhibition of PGF₂-induced contraction in cells isolated from gonadectomized females and pretreated with 17-estradiol for 30 min was not significantly different from that in cells of gonadectomized males (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Effect of pretreatment with 17-estradiol for 30 min on PGF2 (105 M)-induced steady-state changes in cell length and [Ca2+]i in coronary smooth muscle cells isolated from gonadectomized male and female pigs

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Effect of sex hormones on contraction of male porcine coronary smooth muscle cells induced by 105 M PGF2 (A), 10 mM caffeine (B), 105 M carbachol (C), and 51 mM KCl (D). PGF2 and KCl contractions were elicited in the presence of 1 mM extracellular Ca2+. Caffeine and carbachol contractions were elicited in Ca2+-free (2 mM EGTA) Hanks' solution. The cells were pretreated with increasing concentrations of the sex hormones for 30 min, and the cell contraction was measured and presented as the ratio L/Li, where L is the final cell length, and Li is the initial resting cell length. The data points represent the mean ± S.E. of measurements in 7 to 35 cells from 4 to 16 pigs. *, cell contraction in the presence of 17-estradiol is significantly different (p < .05) from that in the presence of progesterone or testosterone.

Both progesterone and testosterone also caused concentration-dependent inhibition of PGF₂-induced contraction, although their inhibitory effects were less than that of 17-estradiol. At a 10⁷ M concentration, progesterone and testosterone significantly decreased the magnitude of PGF₂-induced cell contraction to 0.88 ± 0.02 (n = 12) and 0.89 ± 0.01 (n = 8) of the initial cell length, respectively. The PGF₂-induced contraction in the presence of 10⁷ M progesterone or testosterone was 44.44 or 40.74%, respectively, of maximal PGF₂ contraction in the absence of the hormone. Pretreatment of the cells with the progesterone receptor antagonist RU-486 (10⁶ M) or the testosterone receptor antagonist flutamide (10⁶ M) for 30 min completely abolished the inhibition of PGF₂ contraction by 10⁷ M progesterone or testosterone, respectively. The PGF₂ (10⁵ M) contraction in the presence of progesterone plus RU-486 (0.75 ± 0.02, n = 25) or testosterone plus flutamide (0.74 ± 0.03, n = 16) was not significantly different from that in the absence of the hormone and the antagonist.

We examined the effects of caffeine, a known activator of Ca²⁺ release from the intracellular stores (Leijeten and van Breemen, 1984), on cell contraction. In Ca²⁺-free (2 mM EGTA) Hanks' solution, caffeine (10 mM) caused contraction of coronary smooth muscle cells and decreased cell length to 0.92 ± 0.01 (n = 28) of the initial length. Pretreatment of the cells with increasing concentrations of 17-estradiol, progesterone, or testosterone for 30 min did not significantly inhibit the caffeine-induced cell contraction (Fig. 1B).

The effects of carbachol, an activator of inositol-1,4,5-triphosphate (IP₃) production, and Ca²⁺ release from the intracellular stores (Takuwa et al., 1986) on cell contraction were also examined. In Ca²⁺-free (2 mM EGTA) Hanks' solution, carbachol (10⁵ M) caused contraction of coronary smooth muscle cells and decreased cell length to 0.93 ± 0.01 (n = 30) of the initial length. Pretreatment of the cells with increasing concentrations of 17-estradiol, progesterone, or testosterone for 30 min did not significantly inhibit the carbachol-induced cell contraction (Fig. 1C).

Membrane depolarization by high KCl is known to stimulate Ca²⁺ entry from the extracellular space through voltage-gated Ca²⁺ channels (Bolton, 1979; van Breemen et al., 1979; Nelson et al., 1988). High KCl (51 mM) caused significant contraction of coronary smooth muscle cells and decreased cell length to 0.63 ± 0.02 (n = 21) of the initial length. 17-Estradiol, progesterone, and testosterone caused concentration-dependent inhibition of KCl-induced cell contraction (Fig. 1D). In the presence of 10⁷ M 17-estradiol, the KCl-induced cell contraction was significantly reduced to 0.91 ± 0.01 (n = 10) of the initial cell length. The KCl-induced cell contraction in the presence of 10⁷ M 17-estradiol was 24.32% of maximal KCl-induced contraction in the absence of the hormone. At concentrations of <10⁷ M, progesterone and testosterone did not cause significant inhibition of the KCl-induced contraction. At 10⁷ M, progesterone and testosterone decreased the KCl-induced cell contraction to 0.72 ± 0.02 (n = 24) and 0.73 ± 0.02 (n = 13) of the initial cell length, respectively. The KCl-induced contraction in the presence of 10⁷ M progesterone or testosterone was 75.68 or 72.97%, respectively, of maximal KCl-induced contraction in the absence of the hormone. At concentrations of >10⁷ M, progesterone and testosterone caused further inhibition of the KCl-induced cell contraction (Fig. 1D).

To test whether the sex hormone-induced inhibition of PGF₂-induced cell contraction reflects changes in [Ca²⁺]_i, [Ca²⁺]_i was measured in Fura-2-loaded coronary smooth muscle cells of gonadectomized male pigs. In unstimulated cells incubated in normal Hanks' solution (1 mM Ca²⁺), the basal [Ca²⁺]_i was 81 ± 2 nM (n = 66). Treatment of the cells with 10⁷ M 17-estradiol, progesterone, or testosterone did not cause any significant changes in basal [Ca²⁺]_i. In another group of cells incubated in Hanks' solution (1 mM Ca²⁺), PGF₂ (10⁵ M) caused a biphasic response: an initial rapid (T_1/2 = 34 ± 3 s, n = 37) peak in [Ca²⁺]_i to 316 ± 4 nM (n = 37) followed by a maintained steady-state increase to 297 ± 4 nM (n = 37; Table 1). Application of the sex hormones, on top of the maintained PGF₂-stimulated increases in [Ca²⁺]_i, caused a gradual reduction in [Ca²⁺]_i (Fig. 2). In cells pretreated with 17-estradiol (10⁷ M) for 30 min, the biphasic shape of the PGF₂ (10⁵ M) response was not changed. Under these conditions and in the continuous presence of the hormone, PGF₂ caused an initial rapid (T_1/2 = 31 ± 8 s, n = 8) peak in [Ca²⁺]_i to 324 ± 6 nM (n = 8) that was not significantly different from the PGF₂-induced initial peak [Ca²⁺]_i in the absence of the hormone. Also, under these conditions and in the continuous presence of the hormone, the PGF₂ steady-state increase in [Ca²⁺]_i was reduced to 161 ± 14 nM (n = 8; Table 1), which was not significantly different from the steady-state [Ca²⁺]_i levels observed when PGF₂ was added first and then 17-estradiol (156 ± 8 nM, n = 8). The maintained PGF₂-stimulated [Ca²⁺]_i in the presence of 10⁸ or 10⁷ M 17-estradiol, progesterone, or testosterone was significantly lower than that in the absence of the hormone. Also, the inhibitory effect of 17-estradiol on the PGF₂-stimulated [Ca²⁺]_i was significantly greater than that of progesterone or testosterone (Fig. 3). In the absence of sex hormones, the PGF₂-stimulated steady-state [Ca²⁺]_i in cells of gonadectomized female pigs was not significantly different from that in cells of gonadectomized males (Table 1). Also, the decrease in the PGF₂-stimulated steady-state [Ca²⁺]_i in cells isolated from gonadectomized females and pretreated with 17-estradiol for 30 min was not significantly different from that in cells of gonadectomized males (Table 1).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Effect of sex hormones on PGF2-induced changes in [Ca2+]i in male porcine coronary smooth muscle cells. Cells were stimulated with 105 M PGF2 in Ca2+- and Mg2+- containing Hanks' solution for 5 min. The cells were then treated with the vehicle (A) or 107 M 17-estradiol (B), progesterone (C), or testosterone (D). The figure shows representative traces of measurements in 8 to 37 cells from 4 to 12 pigs.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Effect of different concentrations of sex hormones on PGF2-stimulated increases in [Ca2+]i in male porcine coronary smooth muscle cells. Cells were stimulated with 105 M PGF2 in Ca2+-containing (1 mM) Hanks' solution for 5 min. The cells were then treated with the vehicle or the sex hormone. Data bars represent the mean ± S.E. of measurements in 5 to 37 cells from 4 to 12 pigs. *, the PGF2-stimulated increase in [Ca2+]i in the presence of sex hormone is significantly different (p < .05) from that in the absence of the hormone. a, the PGF2-stimulated increase in [Ca2+]i in the presence of 17-estradiol is significantly different (p < .05) from that in the presence of progesterone or testosterone.

To test whether the sex hormone-induced changes in [Ca²⁺]_i are due to changes in Ca²⁺ release from intracellular stores, we investigated the effects of sex hormones on [Ca²⁺]_i in Ca²⁺-free solution. In Ca²⁺-free (2 mM EGTA) Hanks' solution, the resting [Ca²⁺]_i was significantly reduced to 35 ± 4 nM (n = 20). Treatment of the cells with 10⁷ M 17-estradiol, progesterone, or testosterone did not cause any significant change in resting [Ca²⁺]_i. We also tested whether sex hormones affect agonist-induced [Ca²⁺]_i transients that are triggered by agonist-activated Ca²⁺ release from intracellular stores. In Ca²⁺-free (2 mM EGTA) Hanks' solution, the [Ca²⁺]_i in the presence of PGF₂ (10⁵ M) was 39 ± 4 nM (n = 14), which was not significantly different from that in unstimulated cells. On the other hand, caffeine (10 mM) caused a transient increase in [Ca²⁺]_i to 256 ± 53 nM (n = 25). Pretreatment of the cells with 10⁷ M 17-estradiol, progesterone, or testosterone for 30 min did not significantly change the caffeine-induced increase in [Ca²⁺]_i (Fig. 4, A and B).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Effect of sex hormones on caffeine- and carbachol-induced increase in [Ca2+]i in male porcine coronary smooth muscle cells. The cells were incubated in Ca2+-free (2 mM EGTA) Hanks' solution for 1 min and then stimulated with 10 mM caffeine (A and B) or 105 M carbachol (C), and the changes in [Ca2+]i were observed. Other cells were pretreated with 107 M 17-estradiol, progesterone, or testosterone for 30 min in normal Hanks' solution, incubated in Ca2+-free Hanks' solution for 1 min, and then stimulated with caffeine or carbachol. The data bars represent the mean ± S.E. of measurements in 6 to 25 cells from 4 to 11 pigs.

To investigate whether the sex hormones inhibit Ca²⁺ release from the IP₃-sensitive intracellular Ca²⁺ stores, the effects of the hormones on the carbachol-induced changes in [Ca²⁺]_i in Ca²⁺-free (2 mM EGTA) Hanks' solution were tested. Carbachol (10⁵ M) caused a transient increase in [Ca²⁺]_i to 262 ± 32 nM (n = 10). Pretreatment of the cells with 10⁷ M 17-estradiol, progesterone, or testosterone for 30 min did not significantly change the carbachol-induced increases in [Ca²⁺]_i (Fig. 4C).

The observations that sex hormones reduced the PGF₂-induced increases in [Ca²⁺]_i in the presence of external Ca²⁺ but did not affect the caffeine- or carbachol-induced [Ca²⁺]_i transients in Ca²⁺-free solution suggested that sex hormones possibly inhibit Ca²⁺ entry from the extracellular space. To further investigate whether the sex hormones decrease [Ca²⁺]_i by inhibiting Ca²⁺ entry from the extracellular space, we tested the effects of the hormones on the KCl-induced changes in [Ca²⁺]_i. Membrane depolarization by 51 mM KCl caused a significant and maintained increase in [Ca²⁺]_i to 341 ± 20 nM (n = 29). The application of the sex hormones on top of the KCl-induced increase in [Ca²⁺]_i caused a gradual decrease in [Ca²⁺]_i (Fig. 5). [Ca²⁺]_i measurements in cells pretreated with 17-estradiol (10⁷ M) for 30 min and then stimulated with 51 mM KCl showed that 17-estradiol decreased the KCl-induced increase in [Ca²⁺]_i to 158 ± 12 nM (n = 8), which was not significantly different from the [Ca²⁺]_i levels observed when KCl was added first and then 17-estradiol was added (164 ± 16 nM, n = 8). The steady-state KCl-induced increase in [Ca²⁺]_i in the presence of 10⁸ or 10⁷ M 17-estradiol was significantly lower than that in the absence of the hormone. At 10⁸ M, progesterone and testosterone did not significantly change the KCl-induced [Ca²⁺]_i. On the other hand, at 10⁷ M, progesterone and testosterone caused a significant decrease in the KCl-induced [Ca²⁺]_i (Fig. 6).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Effect of sex hormones on KCl-stimulated [Ca2+]i in male porcine coronary smooth muscle cells. Cells were stimulated with 51 mM KCl solution for 5 min. The cells were then treated with the vehicle (A) or 107 M 17-estradiol (B), progesterone (C), or testosterone (D). The figure shows representative traces of measurements in 8 to 29 cells from 4 to 12 pigs.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effect of different concentrations of sex hormones on 51 mM KCl-stimulated [Ca2+]i in male porcine coronary smooth muscle cells. Cells were stimulated with 51 mM KCl for 5 min and then treated with the vehicle or the sex hormone. The data bars represent the mean ± S.E. of measurements in 8 to 29 cells from 4 to 12 pigs. *, the KCl-stimulated [Ca2+]i in the presence of sex hormone is significantly different (p < .05) from that in the absence of the hormone. a, the KCl-stimulated [Ca2+]i in the presence of 17-estradiol is significantly different (p < .05) from that in the presence of progesterone or testosterone.

	Discussion

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The present study demonstrated that 17-estradiol caused significant relaxation of PGF₂-induced contraction in single coronary smooth muscle cells. These results are in agreement with other studies that have shown that estrogen causes relaxation in isolated rabbit and human coronary arteries (Harder and Coulson, 1979; Jiang et al., 1991; Collins et al., 1993). We found that pretreatment of the cells with17-estradiol for 30 min or longer periods of time (1 h) and then washing the hormone away did not significantly affect the contractile response to PGF₂, suggesting that the continuous presence of the hormone was necessary to elicit inhibitory effect. Although the data suggest low affinity of the hormone for the receptors, these data should be interpreted with caution because the cellular location of the hormone in relation to the receptor cannot be predicted under these conditions. The present study also showed that both progesterone and testosterone caused significant inhibition of coronary smooth muscle contraction, although their inhibitory effects were less than that of 17-estradiol. Our results are consistent with reports that both progesterone and testosterone cause endothelium-independent relaxation of rabbit coronary artery and aorta (Jiang et al., 1992; Yue et al., 1995) and provide direct evidence that both progesterone and testosterone have a potent relaxant effect in single porcine coronary arterial smooth muscle cells. The specificity of the hormone effects in the present study was supported by the observation that the hormone-induced relaxation of agonist-induced contraction was abolished in cells pretreated with selective receptor antagonists to sex hormones. These data also argue against the possibility that the inhibitory effects of the hormones are due to interaction of the hormones with the agonist.

The sex hormone-induced vascular smooth muscle relaxation suggested possible alterations in one of the agonist-induced contraction mechanisms. Agonist-induced activation of vascular smooth muscle is known to activate phospholipase C, an enzyme that causes the breakdown of phosphatidylinositol-4,5-bisphosphate to IP₃ and 1,2-diaclyglycerol (Berridge and Irvine, 1984). IP₃ stimulates Ca²⁺ release from intracellular stores and causes a transient increase in [Ca²⁺]_i (Suematsu et al., 1984), whereas 1,2-diaclyglycerol activates the enzyme protein kinase C (Nishizuka, 1992). In addition, the agonist increases [Ca²⁺]_i by stimulating Ca²⁺ entry from the extracellular space through Ca²⁺ channels.

The present study showed that sex hormones significantly inhibit PGF₂-induced increase in [Ca²⁺]_i. The observation that the sex hormones did not inhibit caffeine- or carbachol-induced contraction or increase in [Ca²⁺]_i in Ca²⁺-free solution suggests that sex hormones do not inhibit smooth muscle contraction by inhibiting Ca²⁺ release from the intracellular stores. On the other hand, our observation that the sex hormones significantly inhibited the high KCl-induced contraction and increase in [Ca²⁺]_i suggests that sex hormones decrease [Ca²⁺]_i in coronary smooth muscle cells by inhibiting Ca²⁺ entry through voltage-gated channels. Other studies have shown that 17-estradiol blocks voltage-gated Ca²⁺ channels in cultured A7r5 cells (Zhang et al., 1994) and rat aortic smooth muscle cells (Nakajima et al., 1995). Although the Ca²⁺ permeability through voltage-gated channels may be different in cultured cells, our present results in freshly isolated coronary smooth muscle cells are still consistent with the findings of these reports.

We investigated whether the sex hormones inhibit the PGF₂- and depolarization-induced contraction by inhibiting the same Ca²⁺ entry pathway. We found that the same concentrations of 17-estradiol caused similar inhibition of PGF₂- and KCl-induced cell contraction and [Ca²⁺]_i, suggesting that regardless of the type of stimulant, 17-estradiol probably inhibits the same Ca²⁺ entry pathway (i.e., voltage-gated Ca²⁺ channels). Interestingly, the progesterone- and testosterone-induced inhibition of PGF₂-induced contraction and [Ca²⁺]_i was significantly greater than the inhibition of the KCl-induced responses. These data suggest that progesterone and testosterone not only inhibit Ca²⁺ entry through voltage-gated channels but also may inhibit additional contractile mechanisms activated by PGF₂, such as Ca²⁺ entry through receptor-operated Ca²⁺ channels (Bolton, 1979; van Breemen et al., 1979) and/or activation of the enzyme protein kinase C (Khalil and Morgan, 1992).

It is important to emphasize the following cautionary statements regarding the above interpretations. First, in the present study, the acute application of sex hormones caused significant relaxation and decreased [Ca²⁺]_i in isolated coronary smooth muscle cells incubated at 22°C. Because the Q₁₀ of the enzyme systems and the membrane phenomena may be affected by the change in temperature, it remains to be investigated whether similar vascular effects also occur under the more physiological in vivo conditions at 37°C and during subacute treatment of animals with various steroids, where the levels of the endogenous sex hormones and the expression of the sex hormone receptors may vary depending on the gender and on the presence or absence of functioning gonads. Second, the present experiments were conducted on coronary smooth muscle cells from gonadectomized pigs. The data suggest that in the absence of circulating levels of sex hormones, the contractile response and [Ca²⁺]_i in coronary smooth muscle cells of male pigs are not significantly different from those in female pigs, and that in the absence of functional gonads, there is no gender difference in the effects of sex hormones on the agonist-induced coronary smooth muscle contraction and [Ca²⁺]_i. However, we cannot generalize that the observed vascular relaxation by sex hormones is the general effect of the hormones on coronary arteries from male or female pigs with intact gonads because the expression of the estrogen, progesterone, or testosterone receptors in the coronary arteries may vary depending on the status of the gonads. Interestingly, a recent study has suggested that estrogen stimulates Ca²⁺ extrusion from coronary arterial smooth muscle cells of gonad-intact, sexually mature female pigs (Prakash et al., 1999). However, this mechanism does not appear to be affected by sex hormones in our present experiments on cells isolated from sexually immature pigs because the caffeine- and carbachol-induced contraction and [Ca²⁺]_i transients were not inhibited by the hormones. Comparison of the vascular effects of sex hormones on coronary arteries from male and female pigs with and without intact gonads should, therefore, represent an important area for future investigation. Several studies have shown that sex hormones bind to specific receptors in a multitude of vascular smooth muscle preparations, including coronary smooth muscle (Harder and Coulson, 1979; McGill and Sheridan, 1981; Ingegno et al., 1988; Losordo et al., 1994; Farhat et al., 1996). It has also largely been recognized that sex steroids diffuse through the plasma membrane and form complexes with specific cytosolic and/or nuclear receptors, which then bind to chromatin and stimulate the expression of a set of genes with specific sex steroid-responsive regulatory element (Horwitz and Horwitz, 1982; Carson-Jurica et al., 1990). On the other hand, recent reports have shown that sex hormones can also bind to cell membranes and induce rapid cellular events within seconds or minutes of application, suggesting a nongenomic action triggered by a signal-generating receptor on the cell surface rather than a gene-activating nuclear steroid-receptor complexes (Landers and Spelsberg, 1992; Farhat et al., 1996). The present study showed that the sex hormones caused rapid decreases in [Ca²⁺]_i in coronary smooth muscle cells. However, we cannot make a definite conclusion regarding whether the sex hormone-induced changes in [Ca²⁺]_i temporally coincide with a distinct subcellular location of the sex hormone-receptor complex at the cell membrane, the cytosol, or the nucleus. The temporal relationship between the spatial subcellular location of the sex hormone-receptor complex and the sex hormone-induced changes in [Ca²⁺]_i should, therefore, represent an important area for future investigation.

In conclusion, the female sex hormones 17-estradiol and progesterone and the male sex hormone testosterone inhibit PGF₂-induced contraction and elevation of [Ca²⁺]_i in coronary smooth muscle cells. Sex hormones do not inhibit Ca²⁺ release from intracellular stores but rather inhibit the increases in [Ca²⁺]_i associated with stimulation of Ca²⁺ entry from the extracellular space. The results suggest that 17-estradiol mainly inhibits Ca²⁺ entry through voltage-gated Ca²⁺ channels, whereas progesterone and testosterone may inhibit Ca²⁺ entry through other types of Ca²⁺ channels or suppress other contractile mechanisms. Further investigations are needed to test the effect of sex hormones on these additional contractile mechanisms.