INTRODUCTION

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ACUTE ADMINISTRATION of estrogens relaxes vascular smooth muscle, causing a reduction in vascular resistance and an increase in blood flow (3, 5, 8, 9, 16, 19, 21, 25, 30). The rapid relaxation of vasomotor tone induced by estrogens is likely mediated through nongenomic effects (for review, see Ref. 2) and probably involves estrogen receptors (2, 11, 20). However, despite considerable evidence for acute, nongenomic effects of estrogen on vascular tissue, the mechanisms underlying these effects are not completely understood.

Estrogen-induced relaxation of vascular smooth muscle involves a reduction in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (15, 19). One demonstrated mechanism for this effect is a decrease in Ca²⁺ influx (8, 9, 14). However, a reduction in [Ca²⁺]_i induced by estrogens may also result from an increase in Ca²⁺ efflux, an inhibition of Ca²⁺ release from the sarcoplasmic reticulum (SR), or an increase in SR Ca²⁺ reuptake. Therefore, the purpose of the present study was to examine whether estrogens [17-estradiol (E₂)] affect Ca²⁺ efflux and SR Ca²⁺ release in coronary artery smooth muscle.

	METHODS

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Animals. Ten sexually mature female Yorkshire pigs (at least 6 mo of age) were obtained from a local supplier. Animals were anesthetized with ketamine (8 mg/kg) and xylazine (12 mg/kg), and their hearts were removed. The three main coronary arteries (right and left circumflex and left anterior descending arteries) were removed and placed in oxygenated modified Krebs-Ringer bicarbonate solution at 4°C. Blood samples were also drawn to verify hormonal status using plasma concentrations of estrogen and progesterone.

Immunocytochemical detection of estrogen receptors. Freshly dissociated coronary artery smooth muscle cells were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer and stained for 2-3 h with a polyclonal antibody to the estrogen receptor (Research Biochemicals; 1:2,000 dilution in phosphate buffer). After staining with the primary antibody was completed, the sections were washed in phosphate buffer and reacted with a 1:1,000 Cy3-conjugated donkey anti-rabbit IgG secondary antibody. The fluorescently labeled coronary artery smooth muscle cells were visualized using a Bio-Rad MRC500 confocal microscope equipped with an argon-krypton laser. The 568-nm laser line was used to excite the Cy3, and the emissions were collected using a 590-nm long-pass filter.

Cell preparation for Ca²⁺ imaging. The procedures for dissociation of single porcine coronary artery smooth muscle cells have been described previously (10). Briefly, the endothelium was removed and the tissue was minced thoroughly in Hanks' balanced salt solution (HBSS) buffered with 10 mM HEPES (pH 7.4; Life Technologies, Gaithersburg, MD). The tissue was incubated first in 20 U/ml papain and 2,000 U/ml DNase (Worthington Biochemical, Freehold, NJ) and subsequently in 1 mg/ml type IV collagenase and 0.1 mg/ml elastase (Worthington). Single cells were released by trituration, centrifugation, and resuspension in minimum essential medium with 10% fetal calf serum. The cells were plated on glass coverslips coated with Cell-Tak (Collaborative Biomedical Products, Bedford, MA) and incubated for 1-2 h in a 5% CO₂ incubation chamber at 37°C. Cells were used within 4 h after dissociation. No time-dependent changes were observed in the gross morphology of the cells that were used for the experimental protocols. Exclusion of the dye trypan blue was used to assess cell viability just after dissociation (>90% of all cells). An anti-smooth muscle myosin antibody (Sigma Immunochemicals, St. Louis, MO) was used to estimate the relative proportion of smooth muscle myocytes (immunoreactive) and fibroblasts (50:1).

Real-time Ca²⁺ imaging. An Odyssey XL real-time confocal system (Noran Instruments, Middleton, WI) attached to the Nikon Diaphot and equipped with an argon-krypton laser was used to visualize fluo 3-loaded coronary artery smooth muscle cells (18). An Olympus ×40/1.3 oil-immersion objective lens was used to visualize the cells. Image size was set to 640 × 480 pixels, and pixel area was calibrated using a stage micrometer (0.063 µm²/pixel). A fixed combination of laser intensity (20% of maximum) and photomultiplier gain (1,700 from a maximum of 4,096) was set a priori to ensure that pixel intensities within regions of interest ranged between 25 and 255 gray levels across different experimental protocols. Dye bleaching was kept to a minimum by maintaining laser intensity below 3 mW. Continued exposure to the laser did not exceed 3 min. With the use of these precautions, dye bleaching was estimated to be <5% over a 3 min period.

Assessment of cell viability. After each experiment, coronary artery smooth muscle cells on at least one coverslip per animal (~80 cells) were evaluated for the exclusion of trypan blue. These cells were exposed to the confocal laser for varying periods of time ranging from <3 min to repeated exposures across a 30-min period. The exclusion of trypan blue in these cells confirmed that laser exposure did not injure the cells. In a subset of these cells (20 cells), the effect of prolonged laser exposure (three 3-min exposure periods separated by 5 min) on baseline [Ca²⁺]_i level was evaluated. Baseline [Ca²⁺]_i was found to vary <5% across this entire period. In another subset of cells (15 cells), the reproducibility of the [Ca²⁺]_i response to 5 mM caffeine was evaluated over a 20-min period. The average coefficient of variation for the [Ca²⁺]_i response to caffeine in these cells was 6.5%.

[Ca²⁺]_i response to endothelin-1. The [Ca²⁺]_i response of coronary artery smooth muscle cells was measured to increasing concentrations of endothelin-1 (0.1, 1, 10, and 100 nM and 1 µM). Because tachyphylaxis is a well-known effect with endothelin-1, multiple-agonist exposures for the same cell were not possible. Instead, sets of coronary artery smooth muscle cells were exposed to only one endothelin-1 concentration, and the average response was determined.

Effect of estrogens on endothelin-1-induced elevation of [Ca²⁺]_i. Cells were exposed to 100 nM endothelin-1, and an elevation in [Ca²⁺]_i was confirmed. When [Ca²⁺]_i had reached a steady-state level (2-3 min), the cells were exposed to solvent (ethanol 10 nM), E₂ (0.1 nM to 10 µM), 17-estradiol (E₂; 10 nM, the biologically inactive form of estradiol), or triamcinolone acetonide (10 nM; a synthetic steroid unrelated to estrogens).

Role of estrogen receptors in [Ca²⁺]_i response to estrogens. To determine whether estrogen receptors are necessary for mediation of the effect of E₂ on [Ca²⁺]_i, coronary artery smooth muscle cells were exposed for 3 min to the estrogen-receptor antagonist ICI-182780 (1 µM; generously provided by Zeneca Pharmaceuticals, Cheshire, UK). Subsequently, the cells were exposed to endothelin-1, and when steady-state [Ca²⁺]_i was reached, the cells were exposed to either E₂ or E₂ (10 nM).

Effect of E₂ on Ca²⁺ efflux. To block Ca²⁺ influx, cells were exposed to zero extracellular Ca²⁺ or nifedipine (100 nM). The cells were then exposed to thapsigargin (1 mM) to block SR Ca²⁺ reuptake (23), which resulted in a gradual increase in [Ca²⁺]_i, plateauing after ~2 min. The cells were then exposed to E₂ in the presence or absence of endothelin-1. To determine whether estrogen receptors mediate the effect of E₂ on Ca²⁺ efflux under these conditions, cells were preexposed to ICI-182780 (1 µM) before endothelin-1 exposure. To confirm that Ca²⁺ efflux was actually involved, experiments were repeated in the presence of lanthanum (1 mM), which nonselectively blocks both Ca²⁺ influx and efflux (28).

Effect of E₂ on SR Ca²⁺ release and reuptake. The response to caffeine (5 mM) was evaluated in the presence or absence of preexposure to E₂ in cells in which Ca²⁺ influx and SR Ca²⁺ reuptake were blocked by nifedipine (or zero extracellular Ca²⁺) and thapsigargin, respectively. Under these conditions, SR Ca²⁺ release was estimated from the peak [Ca²⁺]_i response.

Effect of E₂ on Ca²⁺ influx. Cells were exposed to BAY K 8644 (100 nM, 1 and 10 µM) to induce Ca²⁺ influx. Nifedipine (100 nM) completely blocked the elevation of [Ca²⁺]_i induced by 100 nM and 1 µM BAY K 8644, but it only partially blocked the [Ca²⁺]_i response to 10 µM BAY K 8644. Accordingly, Ca²⁺ influx was induced by 1 µM BAY K 8644 in the presence of E₂.

Statistical analysis. In determining the statistical design for various experiments, the influence of interanimal variability in the [Ca²⁺]_i response to 100 nM endothelin-1 was evaluated for 15 coronary artery smooth muscle cells from each of 10 animals using a one-way ANOVA. Interanimal variability was found to be nonsignificant at P > 0.25. Based on this result, the contribution of interanimal variability was determined to be insignificant, and the pooling of results from coronary artery smooth muscle cells obtained from different animals was justified. However, for each of the experimental protocols assessing the impact of estrogen on the [Ca²⁺]_i response to 100 nM endothelin-1, cells from at least five animals were studied, and cells from any one animal did not represent >30% of the total. In addition, individual coronary artery smooth muscle cells were exposed to only one experimental protocol. The numbers of cells used for each protocol are reported in the results, and statistical analysis (one-way ANOVA) was based on this number. Statistical significance was tested at an 0.05 level. Data are reported as means ± SE. Reductions in [Ca²⁺]_i are reported as percent changes from the maximum [Ca²⁺]_i response.

	RESULTS

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Estrogen levels and estrogen-receptor status. Serum estrogen ranged from 37 to 103 pM and averaged 56 ± 10 pM in all 10 animals. Immunocytochemical staining confirmed the presence of estrogen receptors in coronary artery smooth muscle cells (Fig. 1). Although the immunocytochemical procedure could not distinguish between nuclear and cytosolic estrogen receptors, the higher intensity of fluorescence staining in the center of the cell suggested the presence of nuclear receptors. However, immunoreactivity for cytosolic estrogen receptors distributed throughout the cell was also clearly present.

View larger version (61K): [in this window] [in a new window]   Fig. 1.   Immunocytochemical staining for estrogen receptors in porcine coronary artery smooth muscle cells. Dissociated cells were fixed in paraformaldehyde, stained with an antibody to estrogen receptors, and visualized using a Cy3-conjugated fluorescent secondary antibody. Nonspecific labeling of secondary antibody was determined by omitting primary antibody (A). Immunoreactivity confirmed presence of estrogen receptors in coronary artery smooth muscle cells. Note higher fluorescence intensity around nucleus (B).

[Ca²⁺]_i response to endothelin-1. Basal [Ca²⁺]_i levels were not significantly different among cells and ranged from 120 to 160 nM (131 ± 3 nM; n = 426). At each concentration of endothelin-1, the [Ca²⁺]_i response of 16 cells was determined. Endothelin-1 at 1 and 10 nM caused a slow and sustained increase in [Ca²⁺]_i. In comparison, 100 nM and 1 µM endothelin-1 caused a more rapid increase in [Ca²⁺]_i, reaching an initial peak after 4-30 s but then decreasing slowly over the next 2-3 min to ~70% of the peak value. The [Ca²⁺]_i responses to varying endothelin-1 concentrations at 30 s and 2 min are reported in Table 1.

Effect of estrogens on endothelin-1-induced elevation of [Ca²⁺]_i. The effect of E₂ on the [Ca²⁺]_i response to endothelin-1 was concentration dependent with an ED₅₀ of ~10 nM (Fig. 2A). The effect of 10 nM E₂ on the endothelin-1 dose-response (1, 10, and 100 nM) was determined (Fig. 2B). Although the [Ca²⁺]_i response varied with endothelin-1 concentration, the inhibitory effect of E₂ was proportionately similar across all endothelin-1 concentrations. Because the relative effect of 10 nM E₂ was comparable across endothelin-1 concentrations, fixed concentrations of E₂ (10 nM) and endothelin-1 (100 nM) were used in all subsequent protocols.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Cumulative dose-response curve of 17-estradiol (E2)-induced reduction of endothelin-1-induced intracellular Ca2+ concentration ([Ca2+]i) in coronary artery smooth muscle cells from female pigs. In presence of a fixed concentration of 100 nM endothelin-1, cells were exposed to progressively larger concentrations of E2 (A; n = 15). Effect of IC50 E2 (10 nM) on [Ca2+]i response to different endothelin-1 concentrations was determined in separate sets of cells (B; n = 12 per endothelin-1 concentration). [Ca2+]i response is reported as percentage of maximum response at a given endothelin-1 concentration.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effect of estrogens on endothelin-1-induced elevation of [Ca2+]i in porcine coronary artery smooth muscle cells. Exposure to 100 nM endothelin-1 (A) induced a large elevation of [Ca2+]i, which was reduced by both E2 (B; n = 56) and 17-estradiol (E2; C; n = 53), albeit to different extents. Compared with control, solvent control (ethanol; n = 15) and an unrelated steroid [triamcinolone acetonide (TAA); n = 14] did not decrease [Ca2+]i (D). * Significant difference (P < 0.05) from control.

Role of estrogen receptors in [Ca²⁺]_i response to estrogens. In the presence of ICI-182780, E₂ had no effect on the [Ca²⁺]_i response to endothelin-1 (Fig. 4; n = 51). In contrast, ICI-182780 did not abolish the effect of E₂ on the [Ca²⁺]_i response to endothelin-1 (Fig. 4; n = 32).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Involvement of estrogen receptors in estrogen-induced reduction of [Ca2+]i in porcine coronary artery smooth muscle cells. In presence of estrogen-receptor antagonist ICI-182780 (ICI), E2 did not reduce endothelin-1-induced elevation of [Ca2+]i (A; n = 51), suggesting that estrogen receptors mediate this effect of E2. In contrast, even in presence of ICI-182780, E2 reduced endothelin-1-induced elevation of [Ca2+]i (B; n = 32 cells), suggesting a non-estrogen receptor-mediated effect. C: summary of [Ca2+]i responses. * Significant difference (P < 0.05) from response to E2.

Effect of E₂ on Ca²⁺ efflux. When Ca²⁺ influx was blocked by nifedipine (n = 22) or zero extracellular Ca²⁺ (n = 13; data not shown), inhibition of SR Ca²⁺ reuptake by thapsigargin caused a gradual increase in [Ca²⁺]_i (Fig. 5). Subsequent exposure to E₂ reduced [Ca²⁺]_i by 95.1 ± 3.4% (Fig. 5; P < 0.05 compared with vehicle control). Under conditions of blocked Ca²⁺ influx (nifedipine: n = 32; zero extracellular Ca²⁺: n = 21; data not shown) and SR Ca²⁺ reuptake (thapsigargin), endothelin-1 induced a large increase in [Ca²⁺]_i (Fig. 6). Subsequent exposure to E₂ reduced [Ca²⁺]_i (Fig. 6). This reduction in [Ca²⁺]_i was blocked by ICI-182780 (n = 22) and lanthanum (n = 31) (Fig. 6).

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Effect of E2 on Ca2+ efflux at basal [Ca2+]i levels. In absence of agonist-induced sarcoplasmic reticulum (SR) Ca2+ release, when Ca2+ influx was blocked by nifedipine (or zero extracellular Ca2+; not shown) and SR Ca2+ reuptake was blocked by thapsigargin, basal [Ca2+]i levels slowly increased (A; n = 22). This trend in [Ca2+]i was reversed by E2 (B; n = 22), suggesting enhancement of Ca2+ efflux.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Effect of E2 on Ca2+ efflux after endothelin-1 (ET) activation. When Ca2+ influx and SR Ca2+ reuptake were blocked by nifedipine (Nif; or zero extracellular Ca2+; not shown) and thapsigargin (Tg), respectively, endothelin-1 induced a large, sustained elevation of [Ca2+]i (A; n = 32). Subsequent exposure to E2 reduced [Ca2+]i (B; n = 32). This effect of E2 on [Ca2+]i was inhibited by lanthanum (La; C; n = 31), indicating that E2 enhances Ca2+ efflux. D: summary of [Ca2+]i responses. * Significant difference (P < 0.05) from response to endothelin-1 alone.

Effect of E₂ on SR Ca²⁺ release and reuptake. Preexposure to E₂ had no effect on the amplitude of the initial response to 100 nM and 1 µM endothelin-1. When Ca²⁺ influx and SR Ca²⁺ reuptake were blocked, the peak [Ca²⁺]_i response to caffeine was comparable in the presence (n = 24) and absence (n = 25) of E₂ (Fig. 7). These results suggest that E₂ had no effect on SR Ca²⁺ release.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Effect of E2 on SR Ca2+ release and reuptake. In absence of Ca2+ influx and SR reuptake, the large, transient [Ca2+]i response to caffeine (A; n = 25) was not affected by preexposure to E2 (B; n = 24). In absence of Ca2+ influx and efflux (blocked by nifedipine and lanthanum), endothelin-1 produced a large, sustained elevation of [Ca2+]i (C; n = 14). Rate of decline of [Ca2+]i response to endothelin-1 was not affected by preexposure to E2 (D; n = 15). These data suggest that SR Ca2+ release and reuptake are not substantially affected by estrogens.

Effect of E₂ on Ca²⁺ influx. Exposure to E₂ significantly reduced the Ca²⁺ influx induced by 1 µM BAY K 8644 (Fig. 8). When BK_Ca channels were blocked by iberiotoxin, E₂ still reduced the [Ca²⁺]_i response to endothelin-1 (Fig. 8). However, this reduction in [Ca²⁺]_i was significantly less than that in the absence of iberiotoxin (57.5 ± 4.1 vs. 95.1 ± 3.1%, respectively).

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Effect of E2 on Ca2+ influx. Exposure of coronary artery smooth muscle cells to BAY K 8644 (BayK), an agonist of voltage-gated Ca2+ influx channels, resulted in a slow elevation of [Ca2+]i (A; n = 14). Preexposure to E2 inhibited BAY K 8644-induced elevation of [Ca2+]i (B; n = 14). Exposure to E2 following BAY K 8644 also decreased [Ca2+]i (tracing not shown). Preexposure to iberiotoxin (IBTX), a specific inhibitor of Ca2+-activated K+ (BKCa) channels, only partially blocked the reduction in [Ca2+]i levels induced by E2 under conditions of inhibited SR reuptake and endothelin-1 stimulation (C; n = 16). In the statistical summary (D), control responses are [Ca2+]i elevations by BAY K 8644 alone, or decrease in [Ca2+]i by E2 in the absence of IBTX. *Significant difference (P < 0.05) from the control response.

	DISCUSSION

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In addition to confirming the inhibitory effect of estrogens on Ca²⁺ influx in coronary artery smooth muscle cells, the results of the present study also demonstrated that estrogens decrease [Ca²⁺]_i by enhancing Ca²⁺ efflux via a receptor-mediated mechanism. The conclusion that E₂ enhances Ca²⁺ efflux was supported by three observations: 1) the reduction in [Ca²⁺]_i by E₂ was blocked by the estrogen-receptor antagonist ICI-182780; 2) the E₂-induced reduction in [Ca²⁺]_i was observed even when Ca²⁺ influx and reuptake were blocked; and 3) the E₂-induced reduction in [Ca²⁺]_i was absent when both Ca²⁺ influx and efflux were blocked by nifedipine (and/or zero extracellular Ca²⁺) and lanthanum.

The enhancement of Ca²⁺ efflux by E₂ represents a novel mechanism by which estrogens regulate [Ca²⁺]_i in response to agonist stimulation. As would be expected, the relative enhancement of Ca²⁺ efflux was independent of the level of endothelin-1 stimulation, as indicated by the comparable reduction in [Ca²⁺]_i at different endothelin-1 concentrations, relative to the peak response at each concentration (e.g., ~50% decrease from the peak response with 10 nM E₂). Confirmation of the effect of E₂ on Ca²⁺ efflux was derived primarily from the fact that the response was blocked by lanthanum. However, lanthanum also blocks Ca²⁺ influx, which has been previously demonstrated to be inhibited by E₂. Therefore, we designed studies to block Ca²⁺ influx independent of any inhibition on Ca²⁺ efflux.

In experimental protocols designed to examine the underlying mechanisms of E₂ action, a 100 nM endothelin-1 concentration was selected based on studies by other investigators (24, 28), who found that the initial [Ca²⁺]_i response at similar endothelin-1 concentrations represents SR Ca²⁺ release and that the subsequent response represents a balance between Ca²⁺ influx and efflux. In this regard, the initial peak [Ca²⁺]_i response at 100 nM endothelin-1 observed in our study most likely reflects SR Ca²⁺ release, whereas the lower steady state reflects a balance between Ca²⁺ influx and efflux. In contrast, endothelin-1 concentrations of 10 nM or less elicit only slow, monotonic elevations in [Ca²⁺]_i (also observed in the present study), which most likely reflect Ca²⁺ influx (24). Accordingly, the selection of 100 nM allowed the examination of both SR Ca²⁺ release and Ca²⁺ flux across the cell membrane. In contrast to these measurements, studies using multicellular coronary artery smooth muscle strips as well as single cells have reported a decline in [Ca²⁺]_i to baseline after the initial peak [Ca²⁺]_i response to endothelin-1. However, it must be noted that this decline in multicellular preparations occurred over a considerably longer time period (>10 min) and most likely reflects the ensemble of individual cellular responses.

The serum estrogen levels of ~50 pM are consistent with previous reports in swine of 20-200 pM (7, 26). However, because estrous stage was not controlled in the present study, the 50 pM value most likely reflects an average across different stages. Given the picomolar serum estrogen levels, the 10 nM concentration of E₂ used to examine [Ca²⁺]_i regulation is supraphysiological. It is difficult to directly extrapolate the reduction in [Ca²⁺]_i observed with 10 nM E₂ in single coronary artery smooth muscle cells to the extent of reduction in vascular tone in vivo because, if anything, the estrogen concentration at the tissue is likely to be smaller. However, it must be noted that we observed decreases of 20-30% in [Ca²⁺]_i even at E₂ concentrations between 100 pM and 1 nM. These observations would suggest that circulating estrogen levels do decrease [Ca²⁺]_i to a somewhat smaller extent, which would be consistent with a role of estrogen as a modulator of vascular tone. We opted for a concentration of 10 nM E₂ because it approximates an IC₅₀ and thus provided a greater sensitivity for assessing the effects of various inhibitors and activators and distinguishing between the effects of Ca²⁺ efflux versus other mechanisms of estrogen action. It is difficult to compare the results of the present study, in which 10 nM E₂ was used, with those of previous reports because a wide range of estrogen concentrations have been used in these studies (4, 14, 16, 19, 21a, 29) that are often greater than circulating estrogen levels and considerably higher than that used in the present study. Regardless, it appears that the mechanisms by which estrogen modulates vascular tone are similar, and it is likely that Ca²⁺ efflux plays a role in estrogen-induced reduction of [Ca²⁺]_i even in vivo.

The specificity of the E₂ effect on [Ca²⁺]_i regulation is supported by the fact that the estrogen-receptor antagonist ICI-182780 blocked the [Ca²⁺]_i response to E₂. In addition, compared with E₂, the biologically inactive isomer E₂ at a similar concentration was significantly less potent in reducing agonist-induced elevation of [Ca²⁺]_i, consistent with previous studies (25, 31). The lack of inhibition of the [Ca²⁺]_i response to E₂ by ICI-182780 suggests a nonspecific non-receptor-mediated effect of E₂. However, the mechanisms underlying the effects of E₂ are not clear. Finally, triamcinolone acetonide, a steroid not related to estrogens, did not reduce the endothelin-1-induced elevation of [Ca²⁺]_i in coronary artery smooth muscle cells. This also suggests that the nonspecific effect of E₂ is unlikely to be an experimental artifact.

The relatively rapid changes in [Ca²⁺]_i observed in the present study, occurring over seconds rather than minutes or hours, are clearly not compatible with the classic genomic mechanism for the action of E₂, which involves translocation of receptors to the nucleus and protein synthesis (1, 12). Therefore, the acute effects observed in the present study are nongenomic in nature and are also consistent with previous studies both in vivo (13, 17, 30) and in vitro (5, 8, 9, 19, 21a, 25) (also see Ref. 2 for a review). The existence of cytosolic estrogen receptors and the recent interest in the role of plasma membrane estrogen receptors are also indicative of nongenomic effects. It is possible that these plasma membrane receptors are also involved in [Ca²⁺]_i regulation by estrogens and mediate some of the effects on [Ca²⁺]_i observed in the present study. However, with the use of light microscopy, it was not possible to localize the estrogen receptors to the plasma membrane.

In addition to an enhancing effect on Ca²⁺ efflux, the results of the present study also confirmed previous observations that E₂ inhibits Ca²⁺ influx (14, 19, 22, 29, 31). For example, E₂ inhibited the BAY K 8644-induced elevation of [Ca²⁺]_i. These results of the present study extend previous reports by establishing that Ca²⁺ influx, at least through L-type Ca²⁺ channels, is blocked even by nanomolar concentrations of estrogen, considerably smaller than those used in previous studies. In porcine coronary artery smooth muscle from castrated males, estrogens were shown to activate BK_Ca channels through a cGMP-dependent mechanism, thus indirectly reducing Ca²⁺ influx (29). In the present study, the observation that iberiotoxin (a potent inhibitor of BK_Ca channels) decreased the extent to which E₂ reduced the endothelin-1-induced elevation of [Ca²⁺]_i supports this mechanism.

Also in general agreement with previous studies (14, 31) is the observation that E₂ had no effect on SR Ca²⁺ release. In the present study, E₂ did not affect the [Ca²⁺]_i response to caffeine, an agonist for ryanodine-receptor channels. Endothelin-1 is also known to activate inositol 1,4,5-trisphosphate (IP₃) production and release Ca²⁺ through IP₃-receptor channels (10, 27). In the present study, preexposure to E₂ did not affect the initial [Ca²⁺]_i response to endothelin-1, suggesting that SR Ca²⁺ release through IP₃-receptor channels is also not affected. Indeed, in permeabilized vascular smooth muscle, Kitazawa et al. (14) found that the force response to exogenous IP₃ was unaffected by E₂. Therefore, it is unlikely that SR Ca²⁺ release through either IP₃- or ryanodine-receptor pathways is affected by E₂. However, these observations do not rule out the possibility that estrogens inhibit SR Ca²⁺ release via other agonists and/or signal transduction pathways. For example, Han et al. (4) found that E₂ does inhibit SR Ca²⁺ release induced by thromboxane A₂. Further experimentation is required to distinguish between these potentially differential mechanisms of estrogen action on the SR. In the present study, the lack of an E₂ effect of the rate of [Ca²⁺]_i decline under conditions of blocked Ca²⁺ influx and efflux suggests that SR Ca²⁺ reuptake is also unaffected by estrogens. Thus the predominant site of action for estrogens appears to be the plasma membrane.

In summary, the results of the present study demonstrate that E₂ enhances Ca²⁺ efflux, thereby providing a novel mechanism by which estrogens decrease the [Ca²⁺]_i response to endothelin-1 in coronary artery smooth muscle cells from gonad-intact female pigs. This effect of E₂ requires estrogen receptors.