Introduction

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It is well known that women are less prone to ischemic heart diseases than men and that postmenopausal women with estrogen replacement therapy are less likely to develop ischemic heart diseases (Stampfer et al., 1991). It was reported that in women with coronary heart disease, acute administration of estrogen at physiological concentrations, which was reported to range from 10¹⁰ to 10⁹ M (Abraham et al., 1972; Rosano et al., 1993; Gilligan et al., 1994), reduces myocardial ischemia injury (Rosano et al., 1993, 1997). The observation suggests that estrogen may protect the heart against myocardial ischemia. However, studies on the acute effects of the female hormone on the heart and its mechanisms of action are scarce. A recent study showed that 17-estradiol at 1 nM inhibited the stimulatory action of isoproterenol (Iso), a selective -adrenoceptor agonist, on Ca²⁺ influx via the L-type Ca²⁺ channel in the ventricular myocyte (Meyer et al., 1998). It is therefore likely that this negative modulatory action of estrogen against -adrenergic stimulation on Ca²⁺ influx may lead to reductions in the heart rate and contraction. This may reduce oxygen consumption and produce cardioprotection.

It is well established that -adrenergic stimulation activates the adenylate cyclase (AC), leading to an increased cyclic AMP (cAMP) production, which in turn increases Ca²⁺ influx (Tsien et al., 1986; Ochi et al., 1996) and augmented myocardial ischemia injury. Modulation of the actions of -adrenergic stimulation may result from inhibitions of cAMP production.

This study had three objectives: first, to determine the cardiac actions of estrogen in the isolated rat heart subjected to normal perfusion or myocardial ischemia; second, to delineate the effects of estrogen on cardiac responses to -adrenoceptor stimulation; and third, to study the mechanisms of action by determining the effects of estrogen on cAMP production. In addition, we determined whether the cytosolic estrogen receptor mediated the action of estrogen. We examined the effects of 17-estradiol, the most effective estrogen, on the heart rate and developed pressures during normal perfusion, and on cardiac rhythm during myocardial ischemia/reperfusion in the isolated heart of male rats, which do not have the classical estrogen receptor (Stumpf et al., 1977). We also studied the effect of 17-estradiol on cAMP production, using a competitive binding method. We compared the responses in the presence and absence of Iso in all experiments. In addition, we determined whether tamoxifen, an antagonist of estrogen receptor, blocked the effect of 17-estradiol, using Ca²⁺ current and cAMP accumulation as parameters for the study. To determine the role of the classical estrogen receptor in mediating the acute effects of estrogen, we compared the effects of the female hormone and tamoxifen on Ca²⁺ current in the heart of both male and female rats. The male does not have the cytosolic estrogen receptor (Meyer et al., 1998; Raafat et al., 1999), whereas the female does (Stumpf et al., 1977). The most important finding of this study was that 17-estradiol at physiological concentrations, which itself had no effect, modulates negatively the effects of -adrenergic stimulation on the systolic pressure and heart rate of the isolated heart. The signaling mechanism may involve an unknown membrane receptor (not the cytosolic estrogen receptor), cAMP, and the L-type Ca²⁺ channel.

	Materials and Methods

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Isolated Perfused Heart Preparation. Male Sprague-Dawley rats weighing 190 to 210 g were used in most experiments because cytosolic estrogen receptor was reported to be absent in the ventricular myocyte of male rats (Stumpf et al., 1977), which is suitable for the study of nongenomic effects of estrogen. Female rats weighing 190 to 210 g were also used in one experiment in which the Ca²⁺ current responses to 17-estradiol, Iso, and tamoxifen were determined and compared with those in the male rat. Ovaries were removed 2 weeks before the experiment. Ovariectomy does not cause any loss in estrogen receptor (Raafat et al., 1999). After each rat was decapitated with a guillotine, the heart was removed and mounted on a Langendorff apparatus. It was perfused with a Krebs-Ringer solution, equilibrated with 95% O₂ + 5% CO₂ (pH 7.4-7.5, 34°C) at a constant pressure of 58 mm Hg and a flow rate of 8 to 10 ml min¹ as described previously (Wong et al., 1990). The heart was perfused for 5 min and allowed to stabilize. Any heart that exhibited arrhythmias during this period was discarded. Global ischemia was achieved by completely stopping the perfusion. After ischemia, perfusion was resumed. All drugs were dissolved in Krebs-Ringer solution and administered into the coronary arteries. Electrocardiogram was monitored and recorded with two electrodes hooked to the apex and the aorta, respectively. Developed left ventricular pressures were determined via an intraventricular balloon that was inflated by injecting 0.1 to 0.2 ml of saline to adjust the end-diastolic pressure to 2 mm Hg, which produces a pressure of 150 mm Hg. In this study, the developed pressure was the pressure recorded minus 150 mm Hg.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Representative traces showing the changes in developed pressures (DP), heart rate, and ECG. Hearts were perfused with Krebs-Ringer for 5 min in all groups. In the control group (Cont), hearts were perfused with Krebs-Ringer for 20 min more. In the estrogen-treated group (E), 17-estradiol at 0.1 nM to 1 µM was perfused for 20 min. In the third group (Iso), Krebs-Ringer solution was perfused for another 15 min, followed by Iso at 100 nM for 5 min. The fourth group received both estrogen and Iso (E + Iso). After the initial 5 min of perfusion with Krebs-Ringer solution, 17-estradiol at different concentrations was perfused for 20 min, and Iso at 100 nM was added to the same solution for the last 5 min of that period. At the end of drug treatment, perfusion was stopped, and hearts were subjected to ischemia for 30 min. This was followed by resumption of flow for another 5 min. The first and third groups received Krebs-Ringer solution during reperfusion, whereas the second and fourth groups received 17-estradiol in the same concentrations given before ischemia.

Arrhythmia Scoring System. To quantify the arrhythmias, we used the scoring system of Wong et al. (1990) that adopted the basic principles used by Curtis and Walker (1988). The principles are: 1) ventricular arrhythmias are more severe than atrial arrhythmias, and 2) the severity of arrhythmias is directly related to the incident and duration. Details of the scoring system are as follows: 0, no arrhythmia; 1, occasional premature atrial contraction or atrial-ventricular block; 2, occasional premature ventricular contraction (< 3/min); 3, frequent ventricular contraction ( 3/min); and 4, ventricular tachycardia or ventricular fibrillation. The score of a heart represented the value of the most severe type of arrhythmias exhibited in the first 2 min of reperfusion. A duration of 2 min was used because the most severe arrhythmias occurred within the first 2 min into reperfusion.

Cell Isolation. Single ventricular myocytes were isolated using a collagenase method as described previously (Ochi et al., 1996). Briefly, the heart was perfused by the Langendorff method with the following solutions: normal Tyrode's solution for 5 min, calcium-free Tyrode's solution for 5 min, collagenase solution for 20 to 25 min, and high-K⁺, Ca²⁺-free Kraftebruhe (KB) solution for 10 min. The separated cells were stored in KB solution at 4°C. The normal Tyrode's solution contained NaCl, 135 mM; KCl, 5.4 mM; CaCl₂, 1.8 mM; MgCl₂, 1.0 mM; HEPES-Tris, 5 mM; and glucose, 10 mM (pH 7.4). Calcium-free Tyrode's was prepared by omitting CaCl₂ from the normal Tyrode's solution. Collagenase (type I; Sigma Chemical Co., St. Louis, MO) was added to the Ca²⁺-free solution to a final concentration of 0.6 mg ml¹. KB solution contained KOH, 110 mM; taurine, 10 mM; oxalic acid, 10 mM; glutamic acid, 70 mM; KCl, 25 mM; KH₂PO₄, 10 mM; EGTA-Tris, 0.5 mM; HEPES-Tris, 5 mM; and glucose, 10 mM (pH 7.4).

Ca²⁺ Current Measurement. Experiments were performed on single ventricular myocytes freshly isolated from rat heart. Membrane currents were recorded with the whole-cell patch-clamp technique (Hamill et al., 1981). Patch electrodes were pulled from borosilicate capillary tubes (Modulohm A/S, Herlev, Denmark) and had resistances of 1.5 to 2.5 M when filled with internal solutions. The internal solution was composed of CsCl, 60 mM; aspartic acid, 50 mM; MgCl_2, 1 mM; CaCl_2, 1 mM; EGTA, 11 mM; and HEPES 10 mM, pH 7.4 with CsOH. K₂ATP (5 mM) was added to the solution just before the experiment began. During the experiment, the cells were superfused at 25°C with the bath solution containing CsCl, 4.8 mM; MgCl₂, 2 mM; N-methyl-D-glucamine, 132 mM; CaCl₂, 2 mM; and glucose, 10 mM (pH 7.4). All drugs were added to the bath solution and applied with a gravity-fed system. Unless otherwise stated, 17-estradiol was administered to the outside of the cell. Eight application capillaries that made up the drug array were positioned a few hundred micrometers from the recorded cell. Solution changes were completed within 2 s, as measured in our previous experiments with dyes.

Assay of cAMP. The method for determination of cAMP was described previously (Bian et al., 1998). Briefly, samples containing 3 × 10⁶ to 6 × 10⁶ freshly isolated ventricular myocytes after Ca²⁺ loading were incubated with 17-estradiol for 5 min and followed by addition of forskolin or Iso for another 5 min. At the end of treatment, cAMP was extracted from the samples and stored at 20°C for competitive binding assay. The protein of the samples was determined by the method of Lowry et al. (1951), using BSA as a standard. A cAMP assay system kit (code TRK 432; Amersham International, Buckinghamshire, UK) was used, and its assay protocol was adopted.

Drugs and Chemicals. 17-Estradiol, tamoxifen, Iso, and forskolin were purchased from Sigma. Both 17-estradiol and tamoxifen were first dissolved in dimethyl sulfoxide (DMSO) from Sigma as a concentrated stock (10 mM). The final concentration of DMSO was 0.01%. We found that DMSO concentrations up to 0.01% had no effect on the heart. Iso was dissolved freshly before each experiment. The concentration of 17-estradiol started at 0.1 to 1 nM because this is within the circulating level of estrogen in the body (Smith et al., 1975). Tamoxifen was effective as an estrogen receptor antagonist at the concentration range of 1 nM to 10 µM (Belfort et al., 1996; Song et al., 1996). We chose the concentration of 100 nM in this study. Iso at 100 nM was chosen because this concentration increased significantly all cardiac responses studied. All experiments were performed under a shelter to avoid the effect of light.

Statistical Analysis. Two-way ANOVA was used for the analysis of the difference between two treatments and the drug effect. One-way ANOVA with the Newman-Keuls test was used to test the difference between individual groups of one treatment. A paired Student's t test was used for comparison between responses before and after drug administration. Computer-assisted nonlinear regression analysis for sigmoidal dose-responses (Liang and Molinoff, 1986; Leung et al., 1992) was adopted for the determination of EC₅₀. A difference of P < .05 was considered statistically significant.

	Results

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Effects of 17-Estradiol on Heart Rate in the Isolated Langendorff Perfused Rat Heart. The heart rate remained relatively constant for 20 min when the heart was perfused with the Krebs-Ringer solution. 17-Estradiol at the range of 10 nM to 1 µM administered acutely for 20 min significantly reduced the heart rate in a concentration-dependent manner (Figs. 1 and 2). The heart rate was significantly increased by 100 nM Iso, and the effects were attenuated in a concentration-dependent manner by 17-estradiol at 0.1 nM to 1 µM (Figs. 1 and 2). More importantly, 17-estradiol at 0.1 to 1 nM, which is within the physiological range in the body (Abraham et al., 1972; Rosano et al., 1993; Gilligan et al., 1994) and which had no effect on the heart rate itself, abolished the stimulatory effect of Iso (Fig. 2). The inhibitory effect of the female sex hormone on heart rate was significantly greater in hearts treated with Iso (Fig. 2).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effects of 17-estradiol on the heartbeat in the isolated perfused heart of the male rat, with or without treatment with Iso. Values are presented as mean ± S.E.; n = 6 to 10. **P < .01 versus corresponding values in control (C), using one-way ANOVA; +++P < .001 indicates significant difference between two treatments with and without Iso, using two-way ANOVA; ##P < .01 versus corresponding values in group without Iso treatment. R2 is correlation coefficient of nonlinear regression.

Effects of 17-Estradiol on Developed Pressures in the Isolated Langendorff Perfused Rat Heart. The developed pressures remained constant when the heart was perfused with the Krebs-Ringer solution for 20 min. Acute administration of 17-estradiol did not affect the systolic pressure (Figs. 1 and 3A). However, at 1 nM to 1 µM it decreased the diastolic pressure slightly, but significantly, in a concentration-dependent manner (Figs. 1 and 3B). The mean pressure was reduced slightly, but significantly, only when 17-estradiol was at 10 nM to 1 µM (Fig. 3C). In hearts treated for 5 min with Iso, which increased the developed pressures, 17-estradiol at 1 nM, which had no effect, significantly attenuated the systolic and mean pressures (Figs. 1, 3A, and 3C). The reductions in systolic (Fig. 3A) and mean (Fig. 3C) pressures caused by 17-estradiol at 1 nM to 1 µM were significantly greater in hearts treated with Iso than in hearts not treated with Iso.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effects of 17-estradiol on the systolic (A), diastolic (B), and mean (C) pressures in the isolated perfused heart of the male rat, with or without treatment with Iso. Values are presented as mean ± S.E.; n = 6 to 10. *P < .05, **P < .01 versus corresponding values in control (C), using one-way ANOVA. +++P < .001 indicates significant difference between two treatments, with and without Iso, using two-way ANOVA; #P < .05, ##P < .01 versus corresponding values in group without Iso treatment. R2 is the correlation coefficient of nonlinear regression.

Effects of 17-Estradiol on Arrhythmias Induced by Ischemia and Reperfusion in the Isolated Langendorff Perfused Rat Heart. In agreement with previous findings (Wong et al., 1990; McHugh et al., 1995), myocardial ischemia/reperfusion induced arrhythmias including atrial arrhythmias, ventricular premature contraction, ventricular tachycardia, and ventricular fibrillation, which usually occurred during the first 2 min of reperfusion (Fig. 1). The arrhythmia score in the control group was 3.30 ± 0.26 (n = 10, Fig. 4). 17-Estradiol at 0.1 to 1 µM significantly reduced the severity of arrhythmias (Figs. 1 and 4). In hearts treated with Iso, 17-estradiol at a lower concentration range of 10 nM to 1 µM significantly reduced the severity of arrhythmias increased by the treatment with Iso (Figs. 1 and 4). The arrhythmia scores after the administration of 17-estradiol at 10 and 100 nM and 1 µM were reduced by 1.21, 1.68, and 1.95, respectively, in the group with Iso treatment. These values were greater than the corresponding values of 0.8, 1.13, and 1.3, respectively, in the group without Iso (Fig. 4).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effects of 17-estradiol on arrhythmias induced by ischemia/reperfusion in the isolated heart of the male rat, with or without treatment with Iso. *P < .05, **P .01 versus corresponding values in control (C), using one-way ANOVA; #P < .05 versus corresponding values in group without Iso treatment. R2 is the correlation coefficient of nonlinear regression.

Effects of 17-Estradiol on cAMP Accumulation in Ventricular Myocytes. To determine whether the female hormone may modulate the action of -receptor stimulation via cAMP, we studied the effects of 17-estradiol on the basal cAMP level and forskolin- and Iso-induced accumulation of cAMP. In agreement with the previous observations (Bian et al., 1998; Zhang and Wong, 1998), 10 µM forskolin, an activator of AC, and 100 nM Iso increased the cAMP level significantly after 5 min of incubation. However, 1 nM 17-estradiol, which itself had no direct effect, significantly decreased the forskolin- and Iso-stimulated cAMP accumulation (Fig. 5).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effects of 1 nM 17-estradiol on cAMP production in the ventricular myocyte of the male rat, in the presence of either 10 µM forskolin or 100 nM Iso, and on estrogen receptor blockade by 100 nM tamoxifen. Values are presented as mean ± S.E. Figures in brackets indicate numbers of samples. *P < .05, **P .001 versus vehicle control. ##P < .01 versus corresponding control without either forskolin or Iso.

Ca²⁺ Current Responses to 17-Estradiol with and without Tamoxifen and/or Iso in the Ventricular Myocytes in Male and Ovariectomized Female Rats. To determine the role of the cytosolic estrogen receptor in mediating the acute effects of the female hormone, we also determined the effects of 17-estradiol with and without tamoxifen and/or Iso in the ventricular myocytes of ovariectomized female rats and compared them with effects in the male. As in previous studies (Meyer et al., 1998), we used Ca²⁺ influx as a parameter and showed in our laboratory (Fig. 6A) that 17-estradiol inhibited the Ca²⁺ influx via the L-type Ca²⁺ channel. At 1 nM the female hormone inhibited the Ca²⁺ current in the heart treated with 100 nM Iso, whereas at 100 nM it inhibited the Ca²⁺ current in hearts with and without Iso, indicating that at 100 nM the female hormone has both direct action itself and modulatory action against -adrenergic stimulation. Therefore, we chose this concentration in this study. 17-Estradiol and tamoxifen alone reduced (Fig. 6B), whereas Iso alone increased (Fig. 6C), the Ca²⁺ current in the ventricular myocytes of both male and ovariectomized female rats. Tamoxifen did not change the effects of 17-estradiol in ventricular myocytes with or without Iso treatment in both types of rats (Fig. 6). 17-Estradiol also attenuated the effects in Iso-treated ventricular myocytes (Fig. 6C). The most important observation was that all of the effects were similar in the male and female rats (Fig. 6).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effects of 17-estradiol with or without treatment with tamoxifen and/or Iso on the Ca2+ current in the ventricular myocyte of male and ovariectomized female rats. A, effects of 17-estradiol at 1 and 100 nM in the male rat; B, effects of 100 nM 17-estradiol with or without tamoxifen; C, effects of 100 nM 17-estradiol with or without Iso and tamoxifen. Values and control are presented as mean ± S.E., are percentages of the pretreatment control. Figures in brackets indicate numbers of cells. **P < .01 versus corresponding values in control, using one-way ANOVA; ++P < .01 versus corresponding values in Iso treated groups, using one-way ANOVA. There is no significant difference between male and female, using two-way ANOVA.

	Discussion

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The most interesting observations in this study are: 1) 17-estradiol, the most effective estrogen, at concentrations as low as 0.1 to 1 nM, which are within the circulating level in the body (Abraham et al., 1972; Rosano et al., 1993; Gilligan et al., 1994), reduced the heart rate and developed pressures in the heart treated with Iso, a selective -adrenoceptor agonist; and 2) that the female sex hormone also inhibited the augmented cAMP production on -adrenergic stimulation. These observations indicate that the female hormone at physiological concentrations may negatively modulate the cardiac effects of -adrenoceptor stimulation. It should also be noted that at the same concentrations, the potencies of 17-estradiol on the reduction of heart rate and systolic pressure were significantly greater than the corresponding values in hearts without Iso treatment. The results indicate that the responsiveness of the heart to 17-estradiol was significantly greater when the heart was treated with a -adrenoceptor agonist.

It has been well established that -adrenergic stimulation activates the AC/cAMP pathway, which in turn causes cAMP-dependent phosphorylation of the L-type Ca²⁺ channel protein, leading eventually to an increased Ca²⁺ influx via the L-type Ca²⁺ channel (Tsien et al., 1986; Ochi et al., 1996). In this study we demonstrated for the first time that 1 nM 17-estradiol inhibited significantly the enhanced cAMP production by Iso. Meyer et al. (1998) and this study showed that the female sex hormone inhibited the increased Ca²⁺ influx via the L-type Ca²⁺ channel stimulated by Iso in the heart. The observations indicate that the modulatory action of the female sex hormone is most likely mediated via the AC/cAMP pathway, which in turn affects Ca²⁺ influx via the L-type Ca²⁺ channel. Because 17-estradiol also inhibited the forskolin-stimulated increase in cAMP production, the female hormone may also directly inhibit the AC. In fact 17-estradiol has been reported to inhibit the calmoduline-insensitive AC in the rat pulmonary smooth muscle (Farhat et al., 1996). In addition, 17-estradiol has been shown to inhibit the Ca²⁺ current via a G-protein in the neostriatal neuron (Mermelstein et al., 1996). The signaling mechanisms underlying the interaction between the female hormone and -adrenoceptor in the heart warrant additional study.

In an attempt to determine whether the actions of the female sex hormone were mediated via the estrogen cytosolic receptor, we made use of an estrogen receptor antagonist, tamoxifen. One hundred nanomolar tamoxifen, which itself had no effect on cAMP production, did not change the inhibitory effects of 17-estradiol on forskolin- and Iso-induced augmentation in cAMP accumulation. The observation indicates that the effect of 17-estradiol was not mediated via the estrogen receptor, which is located in the cytoplasm. To determine the role of the estrogen receptor in mediating the acute action of estrogen, we compared the effects of 17-estradiol with and without tamoxifen or Iso in the ventricular myocytes of male and female ovariectomised rats. The female rat possesses the cytosolic estrogen receptor (Meyer et al., 1998; Raafat et al., 1999), whereas the male rat does not (Stumpf et al., 1977). The responses in Ca²⁺ current in the ventricular myocyte to the female hormone, with or without tamoxifen and/or Iso, were similar. The observation indicates that the acute action of estrogen does not involve the cytosolic estrogen receptor. In view of its fast action, it is likely that the female sex hormone may act on a membrane receptor. In support of its action on a membrane receptor, we also found in our preliminary study that 1 nM 17-estradiol was only effective in inhibiting the L-type Ca²⁺ current in cells treated with Iso when it was administered to the outside. This is in keeping with the previous finding in the neostriatal neuron (Mermelstein et al., 1996).

In this study, we found that when 17-estradiol at 10 nM to 1 µM was administered acutely, it reduced concentration dependently the diastolic pressure and heart rate in the isolated perfused rat heart. These findings agree with previous observations (Jiang et al., 1992; Hale et al., 1996; Kim et al., 1996). The failure to observe a cardiac action at physiological concentrations in these studies may be due to the fact that the heart was not subjected to -adrenergic stimulation.

17-Estradiol has been shown to inhibit the Ca²⁺ current in the heart in previous (Jiang et al., 1992; Meyer et al., 1998) and present studies. The inhibition in Ca²⁺ current was accompanied by reductions in developed pressures. The observations suggest that the inhibition of Ca²⁺ influx may be responsible at least partly for the reductions in developed pressures. 17-Estradiol may also inhibit Ca²⁺ current in the S-A node, resulting in a reduction in heart rate. Additional study is needed.

It is of interest to note that 17-estradiol reduced the systolic pressure in hearts treated with Iso, but not in hearts without Iso treatment. This may partially be explained by the observation that the inhibitory effect of 17-estradiol on L-type Ca²⁺ current was much greater in hearts with Iso than the minimal effect in hearts without Iso as shown in this study. The inhibitory effect in the former case is probably large enough to reduce the systolic pressure significantly, whereas in the latter case the effect is just too small to cause any significant reduction in contractility.

In this study we found that 17-estradiol only produced an antiarrhythmic effect at 10 nM in the hearts with Iso and at 100 nM in the hearts without Iso. The effective concentrations were 10 to 100 times higher than those required to reduce the heart rate and developed pressures. The low potency of estradiol against arrhythmias during ischemia/reperfusion may be due to the fact that ischemia/reperfusion-induced arrhythmias result from a multitude of factors. Influx of Ca²⁺, which the female hormone inhibits, is only one of the factors.

Sympathetic influence via the -adrenoceptor is perhaps the most important extrinsic factor in the control of the heart. Thus excessive alterations in sympathetic influence may impair the cardiac function. When the heart is subjected to ischemia, there is an increased release of catecholamines as a result of an increased sympathetic influence. Catecholamines may build up to a micromolar concentration in the myocardium if ischemia lasts for 20 to 40 min (Dart et al., 1984; Schoming, 1990). In addition, there are increases in the number of functionally coupled -adrenoceptors (Mukherjee et al. 1982; Maisel et al., 1985) and responsiveness (Schoming, 1990) in the ischemic myocardium. The build-up of catecholamines and increases in -adrenoceptor and responsiveness during ischemia may increase the workload of the heart unnecessarily and cause life-threatening ventricular arrhythmias. Rosano et al. (1993, 1997) showed that the female sex hormone may protect the heart during ischemia. This can be explained by the fact that 17-estradiol attenuates the excitatory effects of Iso on the heart rate and systolic pressure, thus reducing the unnecessary workload and oxygen consumption of the heart during ischemia when excessive catecholamines are released. This results in cardioprotection. The negative modulation of the -adrenoceptor by the female sex hormone also offers an explanation as to why the cardiac contractility of menopausal women is greater than that of premenopausal women (Pines et al., 1992).

In conclusion, we have provided evidence for the first time that 17-estradiol at physiological concentrations administered acutely affects the heart rate and contraction by modulating negatively the -adrenoceptor. The signaling pathway may include an unknown membrane receptor, AC/cAMP pathway, and the L-type Ca²⁺ channel. Additional study is required to delineate the signaling mechanism in more detail and to characterize the receptor.