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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1034-1040.)
© 1999 American Heart Association, Inc.

Original Contributions

Antagonistic Effects of 17ß-Estradiol, Progesterone, and Testosterone on Ca²⁺ Entry Mechanisms of Coronary Vasoconstriction

Janice K. Crews; Raouf A. Khalil

From the Department of Physiology and Biophysics and The Center for Excellence in Cardiovascular-Renal Research, University of Mississippi Medical Center, Jackson.

	Abstract

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Abstract—The clinical observation that coronary artery disease is more common in men and postmenopausal women than in premenopausal women has suggested cardioprotective effects of female sex hormones including hormone-mediated coronary vasodilation. The purpose of this study was to investigate whether the sex hormone-induced coronary relaxation is caused by inhibition of Ca²⁺ mobilization into coronary smooth muscle. The effects of 17ß-estradiol, progesterone, and testosterone on vascular reactivity and ⁴⁵Ca²⁺ influx were tested in deendothelialized coronary artery strips isolated from castrated male pigs. Prostaglandin F₂ (PGF₂) (10^-5 mol/L) caused significant, maintained contraction of coronary artery strips. Caffeine (25 mmol/L), an activator of Ca²⁺ release from intracellular stores, caused transient contraction in Ca²⁺-free solution whereas membrane depolarization by 96 mmol/L KCl, an activator of Ca²⁺ entry, caused maintained contraction in the presence of external Ca²⁺. The 3 sex hormones caused significant and concentration-dependent relaxation of PGF₂- and 96 mmol/L KCl-induced contractions with 17ß-estradiol being the most effective. The sex hormones did not significantly affect the transient caffeine contraction in Ca²⁺-free solution. In contrast, the sex hormones significantly inhibited the PGF₂- and KCl-induced ⁴⁵Ca²⁺ influx. 17ß-Estradiol caused similar inhibition of PGF₂- and KCl-induced contractions, suggesting inhibition of the same Ca²⁺ entry mechanism. However, progesterone and testosterone caused greater relaxation of PGF₂-induced contraction than of KCl-induced contraction. We conclude that in coronary arteries of castrated male pigs, sex hormones inhibit Ca²⁺ entry from extracellular space but not Ca²⁺ release from intracellular stores. 17ß-Estradiol mainly inhibits Ca²⁺ entry, whereas progesterone and testosterone cause coronary relaxation by inhibiting other mechanisms in addition to Ca²⁺ entry.

Key Words: sex hormones • calcium • coronary • contraction

	Introduction

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Coronary artery disease is one of the most common and costly diseases. Coronary vasospasm and subsequent occlusion are frequently associated with increased cardiovascular risk and may lead to myocardial infraction and death.¹ Clinical data suggest that the incidence of coronary heart disease is greater in men and postmenopausal women compared with premenopausal women. This is believed to be because of putative cardioprotective effects of the female sex hormone estrogen. The cardioprotective effects of estrogen have been explained by several mechanisms, including modification of lipid and carbohydrate metabolism,² modification of the composition of circulating lipoproteins,^{3 4 5} and changes in blood coagulation,⁶ as well as direct cardiovascular protective effects on hemodynamics.⁷ Estrogens are vasodilators. For example, it has been reported that subcutaneous administration of ß-estradiol in female guinea pigs reversibly and significantly lowers both the resting systolic blood pressure and the peak systolic blood pressure induced by a pressor challenge of norepinephrine.⁸ Also intravenous infusion of estrogen in ovariectomized nonpregnant sheep has been shown to cause a significant increase in cardiac output and a decrease in systemic vascular resistance whereas local application of estrogen to the uterine artery only causes uterine vasodilation and increased uterine blood flow.⁹ The vasodilator effects of estrogen have also been observed in normal coronary arteries.¹⁰

The vascular endothelium has been suggested to play a role in mediating the estrogen-induced vasodilation. However, indomethacin does not affect the 17ß-estradiol-induced relaxation in endothelium-intact coronary arteries,¹⁰ indicating that the release of vasodilator prostanoids is not involved in the 17ß-estradiol-induced coronary relaxation in vitro. Furthermore, estrogen causes vasodilation in deendothelialized rabbit coronary artery precontracted by endothelin-1, prostaglandin F₂ (PGF₂), and high-potassium depolarizing solution¹⁰ suggesting that the estrogen-induced inhibition of vascular tone has an endothelium-independent component that involves direct action on vascular smooth muscle.^{5 11 12}

Although several studies have addressed the putative cardiovascular benefits of estrogen, there is little and rather inconsistent information on the vascular effects of other sex hormones. For example, the effects of the other female sex hormone, progesterone, and the male sex hormone, testosterone, on the reactivity of vascular smooth muscle, in general, and coronary smooth muscle, in particular, to various vasoconstrictor agonists have been less clear and have ranged from no effect¹³ or increased vascular reactivity^{14 15} to potent vascular relaxation.¹⁶

Also, vasoactive eicosanoids are metabolites of arachidonic acid synthesized by and released from platelets, white blood cells, and vascular smooth muscle cells in response to tissue injury. Among these eicosanoids, the cyclooxygenase metabolite PGF₂ has a potent vasoconstrictive effect in various vascular beds, including coronary vessels. Although vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasm,^{17 18 19 20} little is known about the modulation of their vasoconstrictive action by sex hormones.

Furthermore, the cellular mechanisms of the sex hormone-induced changes in the contractility of vascular smooth muscle, in general, and coronary smooth muscle, in particular, have not been clearly elucidated. Specifically, the effects of sex hormones on the Ca²⁺ mobilization mechanisms in coronary smooth muscle, namely Ca²⁺ release from the intracellular stores and Ca²⁺ entry from the extracellular space, remain unclear.

The purpose of the present study was the following: (1) To determine whether sex hormones other than estrogen cause coronary vascular relaxation and the magnitude of this relaxation, if any, compared with that induced by estrogen. Therefore, the effects of estrogen and the other major female sex hormone, progesterone, as well as the male sex hormone, testosterone, on the contraction of coronary artery strips induced by the vasoactive eicosanoid PGF₂ were compared. (2) To determine whether sex hormones inhibit Ca²⁺ release from the intracellular stores. Therefore, the effects of sex hormones on caffeine-induced contraction were investigated. (3) To determine whether sex hormones inhibit Ca²⁺ entry from the extracellular space and to investigate the possible Ca²⁺ entry pathways involved. Therefore, the effects of sex hormones on PGF₂- and depolarization-induced Ca²⁺ influx were investigated.

	Methods

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Tissue Preparation
Castrated male Yorkshire pigs (20 to 30 kg) were anesthetized by inhalation of isoflurane (Ohio Medical Products). This animal model was used to minimize the contribution of endogenous sex hormones. The abdominal cavity was opened and the animal was bled by severing the renal artery. The chest cavity was opened and the heart was removed rapidly and immersed in normal Krebs solution. All procedures were performed in accordance with the guidelines of the Animal Care and Use Committee at the University of Mississippi Medical Center and the American Physiological Society. The left anterior descending coronary artery was dissected under a dissection microscope, cleaned of connective tissue, and cut transversely into 3-mm-wide rings. The endothelium was removed by gently rubbing the vessel interior with forceps.

Isometric Tension
Coronary artery rings were cut open into strips. One end of the strip was attached to a glass hook using a thread loop and the other end was connected to a Grass force transducer (FT03, Astro-Med). Strips were stretched to 2 g of tension and allowed to equilibrate for 1 hour in a water-jacketed, temperature-controlled organ bath filled with 50 mL Krebs solution continuously bubbled with 95% O₂/5% CO₂ at 37°C. Preliminary experiments with coronary artery strips equilibrated to 0.5, 1, 1.5, and 2 g of resting tension showed that maximal 96 mmol/L KCl-induced contraction was obtained in tissues adjusted to 2 g of resting tension. This resting tension was equivalent to stretching the tissue to approximately 1.5 times its original length. Further increases in resting tension did not significantly change the maximal contraction to 96 mmol/L KCl. The changes in isometric tension were recorded on a Grass polygraph (model 7D, Astro-Med). Removal of the endothelium was routinely verified by the absence of acetylcholine (10^-6 mol/L)-induced vasorelaxation in coronary strips precontracted with PGF₂ (3x10^-7 mol/L).

Three different protocols were followed in this study. In the first protocol, PGF₂ (10^-5 mol/L) was added to the Krebs solution. When the contraction reached a plateau, the tissue was rinsed with Krebs solution 3 times for a duration of 10 minutes each. The whole procedure of contraction and washing was repeated 2 times. PGF₂ (10^-5 mol/L) was then added to the Krebs solution to induce a maximal control contraction. The sex hormones 17ß-estradiol, progesterone, and testosterone were added individually, 1 hormone per chamber. One chamber was used as a control in which the tissue was treated with the vehicle. The hormones were added cumulatively at concentrations ranging from 10^-7 to 10^-4 mol/L, and changes in isometric tension were recorded and presented as percentage of the maximal PGF₂-induced contraction. For each hormone concentration, the effect of the hormone on contraction was allowed to reach a plateau before the next hormone concentration was added.

In the second protocol, the bathing solution was changed to 96 mmol/L KCl to induce contraction. The whole procedure of contraction and washing was repeated 2 times. The bathing solution was changed to 96 mmol/L KCl to induce a maximal control contraction. The sex hormones were added individually, 1 hormone per chamber. The hormones were added cumulatively at concentrations ranging from 10^-7 to 10^-4 mol/L, and changes in isometric tension were recorded and presented as percentage of the maximal 96 mmol/L KCl contraction.

In the third protocol, the tissues were stimulated with 96 mmol/L KCl to induce contraction. Once the KCl contraction reached a plateau, the tissue was rinsed with Krebs solution 3 times for a duration of 10 minutes each. The whole procedure of contraction and washing was repeated 2 times. The tissues were incubated in Ca²⁺-free (2 mmol/L EGTA) Krebs for 5 minutes, and then stimulated with caffeine (25 mmol/L) for a duration of 2 minutes or until the transient contraction returned to the baseline. The bathing solution was then changed to normal Krebs (2.5 mmol/L Ca²⁺) solution, and the tissue was allowed to equilibrate for 1 hour to replenish the intracellular Ca²⁺ stores. After the basal tone reached a steady-state the sex hormones (10^-5 mol/L) were added, 1 hormone per chamber, for a duration of 1 hour. One chamber was used as a control in which the tissue was treated with the vehicle. After 1 hour the Krebs solution was replaced with Ca²⁺-free (2 mmol/L EGTA) Krebs in the continuous presence of the hormone (10^-5 mol/L) or the vehicle. The tissues were incubated in the Ca²⁺-free (2 mmol/L EGTA) Krebs for 5 minutes, and then stimulated with caffeine (25 mmol/L) to elicit a second caffeine contraction.

⁴⁵Ca²⁺ Influx
Coronary artery strips were incubated in normal Krebs solution for 1 hour in preparation for measurement of Ca²⁺ influx as previously described.²¹ The strips were stimulated with 10^-5 mol/L PGF₂ or 96 mmol/L KCl for 30 minutes, and then transferred to PGF₂ or 96 mmol/L KCl solution supplemented with 10^-5 mol/L or 6x10^-5 mol/L 17ß-estradiol, progesterone, or testosterone, or the vehicle, for 1 hour. The incubation times were determined by the time the sex hormone effect on contraction reached a steady-state. The tissues were then transferred to the respective radioactive ⁴⁵Ca²⁺-labeled solution (specific activity, 2 µCi/mL; ICN Radiochemical) for 90 seconds. Preliminary experiments showed that the relationship between ⁴⁵Ca²⁺ uptake versus time is linear during 15-, 30-, 60-, and 90-second exposures to ⁴⁵Ca²⁺-labeled solution. The tissues were transferred to ice-cold Ca²⁺-free (2 mmol/L EGTA) Krebs for 45 minutes to quench extracellular ⁴⁵Ca²⁺ label. The tissues were weighed and placed in 2 mL hypotonic (5 mmol/L) EDTA for 24 hours at 4°C to disrupt the cell membranes and release the intracellular content of ⁴⁵Ca²⁺. The next day, 4 mL of Ecolite scintillation cocktail was added, and the samples were counted in a scintillation counter (LS 6500, Beckman Instruments).

Solutions
Normal Krebs solution contained, in mmol/L: NaCl, 120; KCl, 5.9; NaHCO₃, 25; NaH₂PO₄, 1.2; dextrose, 11.5; MgCl₂, 1.2; CaCl₂, 2.5. The solution was bubbled with 95% O₂/5% CO₂ to adjust the pH to 7.4. For the Ca²⁺-free Krebs solution CaCl₂ was omitted and 2 mmol/L EGTA was added. The high-KCl depolarizing solution was prepared as Krebs solution but with equimolar substitution of NaCl with KCl.

Drugs and Chemicals
Stock solution of PGF₂ was prepared as 10^-2 mol/L in distilled water. Caffeine was prepared as 25 mmol/L in Ca²⁺-free (2 mmol/L EGTA) Krebs solution. Stock solution of 17ß-estradiol (2,3,5[10]-estratriene-3,17ß-diol; Sigma Chemical Co) was prepared as 5x10^-2 mol/L in 100% ethyl alcohol. Stock solutions of progesterone (4-pregnene-3,20-dione; Sigma) and testosterone (4-androsten-17ß-ol-3-one; Sigma) were prepared as 10^-1 mol/L in 100% ethyl alcohol. Diluted 17ß-estradiol, progesterone, and testosterone solutions were also made in 100% ethyl alcohol. All other chemicals were of reagent grade or better.

Statistical Analysis
The developed force in the presence of the hormone was presented as the percentage of maximal PGF₂-, caffeine-, or KCl-induced force in the absence of the hormone. Data were analyzed and expressed as mean±SEM. Data were compared using 1-way ANOVA with Scheffe test and unpaired Student's t test. Differences <0.05 were considered statistically significant.

	Results

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In normal Krebs (2.5 mmol/L Ca²⁺) solution PGF₂ (10^-5 moll/L) caused a significant contractile response of coronary artery strips that was 48.87±9.21% (n=7) of control 96 mmol/L KCl contraction and was maintained for at least 4 hours. The 3 sex hormones 17ß-estradiol, progesterone, and testosterone caused significant relaxation of the PGF₂-induced contraction (Figure 1). The sex hormone-induced relaxation of PGF₂-induced contraction was concentration-dependent (Figure 2). 17ß-Estradiol (IC₅₀=8x10^-6 mol/L) was more effective at inducing coronary relaxation than progesterone (IC₅₀=2.7x10^-5 mol/L) or testosterone (IC₅₀=2.3x10^-5 mol/L). Similar concentrations of the nonsex steroid hormones 11-deoxycorticosterone and deoxycorticosterone acetate did not cause any significant inhibition of PGF₂-induced contraction.

View larger version (12K): [in this window] [in a new window]   Figure 1. Effect of sex hormones on PGF2-induced contraction in porcine coronary artery. Coronary artery strips were stimulated with PGF2 (10-5 mol/L). When the PGF2-induced contraction reached a steady-state the tissue was treated with the vehicle (A) or with 10-5 mol/L 17ß-estradiol (B), progesterone (C), or testosterone (D), and the changes in the magnitude of the maximal PGF2-induced contraction were observed. The figure shows representative traces of measurements in 4 coronary arterial strips from 1 of 8 pigs.

View larger version (17K): [in this window] [in a new window]   Figure 2. Effect of sex hormones on steady-state PGF2 contraction in coronary artery strips. Inhibition was measured at increasing concentrations of the hormones and presented as percentage of initial maximal PGF2-induced contraction. Data represent mean±SEM of measurements in individual coronary artery strips from 8 pigs. *Indicates inhibition of PGF2-induced contraction by 17ß-estradiol is significantly different (P<0.05) from that by progesterone or testosterone.

Agonist-induced contraction of vascular smooth muscle may involve Ca²⁺ release from the intracellular stores or Ca²⁺ entry from the extracellular space.²² Agonist-induced contraction in Ca²⁺-free solution has been used to measure the Ca²⁺ release component of smooth muscle contraction and has been shown to be rapid and transient.²³ In Ca²⁺-free (2 mmol/L EGTA) Krebs, PGF₂ caused a small but maintained contraction that reached 5.52±1.07% (n=7) of control 96 mmol/L KCl contraction. The observation that the PGF₂-induced contraction in Ca²⁺-free Krebs was maintained suggested that it might not solely reflect Ca²⁺ release from the intracellular stores. In contrast, caffeine is known to stimulate Ca²⁺ release from the intracellular stores and to cause transient contraction in vascular smooth muscle.²³ In the control experiments, caffeine (25 mmol/L) caused a small transient contraction in Ca²⁺-free (2 mmol/L EGTA) solution that was 7.38±0.70% (n=22) of the control 96 mmol/L KCl contraction. The tissues were washed 3 times in normal Krebs (2.5 mmol/L Ca²⁺) solution to replenish the intracellular Ca²⁺ stores. Under these conditions a second caffeine contraction in Ca²⁺-free (2 mmol/L EGTA) was 97.4±7.32% (n=4) of the first caffeine contraction (Figure 3). The caffeine response in the tissues pretreated with the sex hormones was not significantly different from that in the control tissues treated with the vehicle (Figure 3).

View larger version (15K): [in this window] [in a new window]   Figure 3. Effect of sex hormones on transient caffeine (25 mmol/L) contraction in coronary artery strips. The first transient caffeine contraction was measured in untreated tissues incubated in Ca2+-free (2 mmol/L EGTA) Krebs. The tissues were transferred to normal Krebs (2.5 mmol/L Ca2+) and treated with the sex hormones (10-5 mol/L) or the vehicle for 1 hour, then a second transient caffeine contraction was elicited in Ca2+-free (2 mmol/L EGTA) Krebs and presented as percentage of the first caffeine contraction. Data represent mean±SEM of measurements in individual coronary artery strips from 4 to 6 pigs. Estr indicates 17ß-estradiol; Prog, progesterone; and Test, testosterone.

To test whether the sex hormones inhibit coronary artery contraction by inhibiting Ca²⁺ entry into the coronary smooth muscle, we tested the effect of the 3 sex hormones on PGF₂-induced ⁴⁵Ca²⁺ influx. In unstimulated coronary artery the basal ⁴⁵Ca²⁺ influx was 11.76±0.57 µmol/kg/min (n=10). PGF₂ (10^-5 mol/L) caused a significant (P=0.049) increase in Ca²⁺ influx to 28.27±6.64 µmol · kg^-1 · min^-1 (n=14). The 3 sex hormones significantly reduced the PGF₂-induced Ca²⁺ entry (Figure 4).

View larger version (13K): [in this window] [in a new window]   Figure 4. Effect of sex hormones (10-5 mol/L) on PGF2-induced 45Ca2+ influx in coronary artery strips. Data represent mean±SEM of measurements in individual coronary artery strips from 14 to 15 pigs. *Indicates PGF2-induced 45Ca2+ influx in the presence of sex hormone is significantly different (P<0.05) from that in the absence of sex hormones.

We investigated the possible Ca²⁺-entry pathways that might be modulated during sex hormone-induced inhibition of coronary artery contraction. Membrane depolarization by high KCl solution is known to stimulate Ca²⁺ entry through voltage-gated Ca²⁺ channels.²² Figure 5 shows that the 3 sex hormones caused significant relaxation of 96 mmol/L KCl-induced contraction with 17ß-estradiol being more effective than progesterone or testosterone.

View larger version (15K): [in this window] [in a new window]   Figure 5. Effect of sex hormones on steady-state 96 mmol/L KCl contraction in coronary artery strips. Inhibition was measured at increasing concentrations of the hormones and presented as percentage of the initial maximal 96 mmol/L KCl-induced contraction. Data represent mean±SEM of measurements in individual coronary artery strips from 6 pigs. *Indicates inhibition of 96 mmol/L KCl contraction by 17ß-estradiol is significantly different (P<0.05) from that by progesterone or testosterone.

To further investigate the effects of sex hormones on Ca²⁺ entry through voltage-gated Ca²⁺ channels, we measured the effect of the sex hormones on the ⁴⁵Ca²⁺ influx induced by 96 mmol/L KCl depolarizing solution (Figure 6). The 96 mmol/L KCl-induced ⁴⁵Ca²⁺ influx in tissues treated with the vehicle was 32.93±4.78 µmol · kg^-1 · min^-1 (n=15). In tissues treated with 17ß-estradiol (10^-5 mol/L) the 96 mmol/L KCl-induced ⁴⁵Ca²⁺ influx was significantly (P=0.033) reduced to 19.03±2.04 µmol · kg^-1 · min^-1 (n=10). In contrast, the 96 mmol/L KCl-induced Ca²⁺ influx in the presence of 10^-5 mol/L progesterone and testosterone was slightly but insignificantly reduced (Figure 6A). However, when the tissues were treated with a higher concentration (6x10^-5 mol/L) of 17ß-estradiol, progesterone, and testosterone, the 96 mmol/L KCl-induced ⁴⁵Ca²⁺ influx was significantly reduced (Figure 6B).

View larger version (15K): [in this window] [in a new window]   Figure 6. Effect of sex hormones at 10-5 mol/L (A) and 6x10-5 mol/L (B) concentration on 96 mmol/L KCl-induced 45Ca2+ influx in coronary artery strips. Data represent mean±SEM of measurements in individual coronary artery strips from 10 to 25 pigs. *Indicates 96 mmol/L KCl-induced 45Ca2+ influx in the presence of sex hormone is significantly different (P<0.05) from that in the absence of sex hormones.

To investigate whether the sex hormones inhibit the PGF₂- and depolarization-induced coronary artery contractions by inhibiting the same Ca²⁺-entry mechanism, we compared the PGF₂-induced contraction with that induced by 96 mmol/L KCl in the presence of increasing concentrations of each of the 3 sex hormones (Figure 7). If the sex hormones inhibit the same Ca²⁺-entry mechanism, we would expect no significant difference in the hormone-induced inhibition of the PGF₂- and KCl-induced contractions. As shown in Figure 7A, there was no significant difference in the relaxation of PGF₂- and KCl-induced contractions by 17ß-estradiol. In contrast, increasing concentrations of progesterone (Figure 7B) or testosterone (Figure 7C) caused significantly greater inhibition of the PGF₂-induced contraction compared with the KCl-induced contraction.

View larger version (20K): [in this window] [in a new window]   Figure 7. Effect of individual sex hormones on the initial maximal steady-state contraction induced by either 10-5 mol/L PGF2 (open symbols) or 96 mmol/L KCl (closed symbols) in coronary artery strips. Inhibition was measured at increasing concentrations of 17ß-estradiol (A), progesterone (B), or testosterone (C), and presented as percentage of the initial maximal contraction to either PFG2 or 96 mmol/L KCl. Data represent mean±SEM of measurements in individual coronary artery strips from 6 to 8 pigs. *Indicates inhibition of 96 mmol/L KCl-induced contraction by a specific concentration of sex hormone is significantly different (P<0.05) from inhibition of PGF2-induced contraction by the same concentration of the same sex hormone.

	Discussion

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The present study demonstrates that (1) both female and male sex hormones cause relaxation of PGF₂- and depolarization-induced contractions of coronary artery with 17ß-estradiol being the most effective, (2) sex hormones do not inhibit caffeine-induced transient contraction of coronary artery, (3) sex hormones inhibit both PGF₂- and depolarization-induced Ca²⁺ entry, and (4) 17ß-estradiol causes similar inhibition of PGF₂- and depolarization-induced coronary artery contractions, whereas progesterone and testosterone cause greater inhibition of the PGF₂-induced contraction than the depolarization-induced contraction.

The present study showed that 17ß-estradiol caused significant relaxation of PGF₂-induced contraction of coronary artery smooth muscle. These results are consistent with the clinical data that suggested cardiovascular protective effects of estrogen.^{24 25 26} The results are also in agreement with experimental data that have shown that estrogen causes vascular relaxation in constricted blood vessels such as rabbit coronary artery^{10 12} and human coronary artery.²⁷

We have also found that both progesterone and testosterone cause significant relaxation of the coronary artery, although the inhibitory effect of these hormones was less potent than that of 17ß-estradiol. Our results with progesterone are different from other reports, which have shown that progesterone induces a negative or opposing effect on blood flow and vasodilation and a minimal effect on canine coronary artery relaxation.¹³ Other studies, however, have shown that progesterone induces endothelium-independent relaxation of rabbit coronary artery.²⁸ The cause of the difference between the results is not clear but may be related to differences in experimental animal species. There have also been inconsistent reports on the effects of testosterone on vascular reactivity. Several studies have reported that testosterone enhances the pressor response to norepinephrine in spinal cat¹⁴ and increases the vascular reactivity to norepinephrine in the perfused hindlimb of dogs.¹⁵ Other studies have shown that testosterone has a potent vasorelaxant effect in the rabbit coronary artery and aorta.¹⁶ The present results provide evidence that testosterone has a potent vasorelaxant effect in porcine coronary artery.

One purpose of the present study was to investigate the effects of sex hormone on Ca²⁺-mobilization mechanisms in coronary smooth muscle, specifically Ca²⁺ release from the intracellular stores and Ca²⁺ entry from the extracellular space. The present results showed that 17ß-estradiol, progesterone, and testosterone did not cause any significant inhibition of the transient caffeine-induced contraction. These results suggest that the vasorelaxant mechanism used by the sex hormones may not involve inhibition of releasable intracellular Ca²⁺ stores. However, the results should be interpreted with caution because the conclusion was based on the experiments with caffeine.

We found that the sex hormones caused significant inhibition of PGF₂-induced Ca²⁺ entry into coronary arterial smooth muscle. Agonist-stimulated Ca²⁺ influx could be through voltage-gated and receptor-operated Ca²⁺ channels.^{29 30} The present results showed that each of the 3 sex hormones caused significant inhibition of the depolarization-induced contraction of the coronary artery. We also found that the sex hormones caused significant inhibition of depolarization-induced Ca²⁺ influx. These results provide the first evidence that sex hormones inhibit coronary artery contraction by inhibiting Ca²⁺ entry through voltage-gated Ca²⁺ channels into coronary smooth muscle. To our knowledge, 1 study has shown that estrogen blocks voltage-gated Ca²⁺ channels in cultured A7r5 cells.³¹ Although the Ca²⁺ permeability through voltage-gated channels may be different in cultured cells, our present results in coronary smooth muscle are still consistent with the findings of this report and should represent an important area for future electrophysiology investigations.

Finally, we investigated whether the sex hormones inhibit PGF₂- and depolarization-induced coronary smooth muscle contractions by inhibiting the same Ca²⁺-entry pathway. We did not find a significant difference between the 17ß-estradiol-induced relaxation of PGF₂- and KCl-induced contractions, suggesting that, regardless of the type of stimulant, estrogen probably inhibits the same Ca²⁺-entry mechanism, namely Ca²⁺ entry through voltage-gated Ca²⁺ channels. However, these findings should be interpreted with caution because estrogens may be equally potent in inhibiting Ca²⁺ entry through voltage-gated Ca²⁺ channels as well as other types of excitable Ca²⁺ channels. Interestingly, the progesterone- and testosterone-induced relaxation of PGF₂- induced contraction was significantly greater than the relaxation of KCl-induced contraction. This suggests that progesterone and testosterone not only inhibit Ca²⁺ entry through voltage-gated channels but may also inhibit other mechanisms activated by PGF₂. These possible mechanisms may include the following. (1) PGF₂ may stimulate Ca²⁺ entry through other types of Ca²⁺ channels, for example the receptor-operated Ca²⁺ channels.^{29 30} If this is the case, then progesterone and testosterone are probably more effective at inhibiting Ca²⁺ entry through receptor-operated Ca²⁺ channels than through voltage-gated Ca²⁺ channels. This is supported by the present observation that relatively lower concentrations (10^-5 mol/L) of progesterone or testosterone were required to significantly inhibit the PGF₂-induced Ca²⁺ influx compared with the relatively greater hormone concentrations required to significantly inhibit the depolarization-induced Ca²⁺ influx. (2) PGF₂ may stimulate other mechanisms in addition to stimulation of Ca²⁺ entry from extracellular space. For example, PGF₂ may activate the enzyme protein kinase C by increasing the formation of diacylglycerol.³² If this is the case, then progesterone and testosterone may be acting by inhibiting these additional contractile mechanisms.

It is important to note that the present experiments were conducted on coronary arteries from castrated male pigs. Therefore, we cannot generalize that the observed vascular relaxation by sex hormones is the general effect of the hormones on coronary arteries from either males with intact gonads or females independent of their hormonal status because expression of estrogen, progesterone, or testosterone receptors in coronary arteries may vary depending on sex and the status of the gonads. 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 interesting area for future investigation. Also, in the present study, micromolar concentrations of sex hormones caused significant vascular relaxation and decreased Ca²⁺ entry in isolated coronary artery strips from castrated male pigs. It remains to be investigated whether similar vascular effects also occur under the more physiological in vivo conditions in which levels of the sex hormones and expression of the sex hormone receptors may vary depending on sex and the presence or absence of functioning gonads.

In conclusion, both the female sex hormones, estrogen and progesterone, and the male sex hormone, testosterone, cause significant relaxation of PGF₂- and depolarization-induced contractions of coronary arteries of castrated male pigs, with estrogen being the most effective. Sex hormones inhibit Ca²⁺ entry into coronary smooth muscle, but not Ca²⁺ release from intracellular stores. The results suggest that estrogen 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 in addition to Ca²⁺ entry. Further studies are needed to investigate the effects of sex hormones on these possible additional contractile mechanisms.

	Acknowledgments

 
This work was supported by grants from the American Health Assistance Foundation, the American Heart Association (Grant-in-Aid, Mississippi Affiliate), and the National Institutes of Health (HL-52696 and HL-51971).

	Footnotes

 
Reprint requests to Raouf A. Khalil, MD, PhD, Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 N State St, Jackson, MS 39216.

Received July 21, 1998; accepted October 5, 1998.

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