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(Circulation. 1995;91:1154-1160.)
© 1995 American Heart Association, Inc.

Articles

Testosterone Relaxes Rabbit Coronary Arteries and Aorta

Ping Yue, MD, PhD; Kanu Chatterjee, MB, FRCP, FACC; Carolyn Beale, BSc; Philip A. Poole-Wilson, MD, FRCP, FACC; Peter Collins, MD, FRCP, FACC

From the Department of Cardiac Medicine, National Heart and Lung Institute, London, and the Cardiovascular Research Institute (K.C.), University of California, San Francisco.

Correspondence to Dr Peter Collins, Department of Cardiac Medicine, National Heart and Lung Institute, London SW3 6LY, UK.

	Abstract

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Background Until menopause, women appear to be protected from coronary heart disease. Evidence suggests that estrogen may play a role in the protection of the cardiovascular system by exerting a beneficial effect on risk factors such as cholesterol metabolism and by a direct effect on the coronary arteries. To date there has been no evidence linking testosterone with the occurrence of coronary heart disease. Testosterone may affect the cardiovascular system directly, thus partially explaining the difference in the incidence of coronary artery disease in men and premenopausal women. The purpose of this study was to assess the direct effect of testosterone and a number of testosterone analogues on rabbit coronary arteries and aorta in vitro.

Methods and Results Rings of coronary artery and aorta of adult male or nonpregnant female New Zealand White rabbits were suspended in organ baths containing Krebs solution; isometric tension then was measured. The response to testosterone was investigated in prostaglandin F₂ (PGF₂)- and KCl-contracted rings. The effects of endothelium and nitric oxide synthase, prostaglandin synthetase, and guanylate cyclase inhibition on testosterone-induced relaxation were investigated. The effects of ATP-sensitive potassium channels and potassium conductance were also assessed. Relaxing responses in the presence of aromatase inhibition and testosterone receptor blockade were performed. The relaxing responses to the testosterone analogues etiocholan-3ß-ol-17-one, epiandrosterone, 17ß-hydroxy-5-androst-1-en-3-one, androst-16-en-3-ol, and testosterone enanthanate were measured. Testosterone relaxed rabbit coronary arteries and aorta. There was no significant difference between the relaxation effect of testosterone with or without endothelium. Similar results were obtained from male and nonpregnant female rabbits. The relaxing response of testosterone in the coronary artery was significantly greater than in the aorta. The relaxing response of testosterone in the coronary artery was significantly reduced by the potassium channel inhibitor barium chloride but not by the ATP-sensitive potassium channel inhibitor glibenclamide. The relaxing response to testosterone was greater in PGF₂-contracted rings compared with KCl-contracted rings. Inhibitors of nitric oxide synthase, prostaglandin synthetase, and guanylate cyclase did not affect relaxation induced by testosterone. Inhibition of aromatase and testosterone receptors did not affect relaxation. Testosterone did not shift the rabbit coronary arterial calcium concentration–dependent contraction curves, whereas verapamil did. There were, however, significant differences in the relaxing response to testosterone compared with testosterone analogues. Testosterone was the most potent relaxing agent, suggesting that there may be a structure-function relation in the relaxing response.

Conclusions Testosterone induces endothelium-independent relaxation in isolated rabbit coronary artery and aorta, which is neither mediated by prostaglandin I₂ or cyclic GMP. Potassium conductance and potassium channels but not ATP-sensitive potassium channels may be involved partially in the mechanism of testosterone-induced relaxation. The in vitro relaxation is independent of sex and of a classic receptor. The coronary artery is significantly more sensitive to relaxation by testosterone than the aorta. Testosterone is a more potent relaxing agent of rabbit coronary artery than other testosterone analogues.

Key Words: hormones • arteries • endothelium • aorta

	Introduction

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The most important benefit of hormone replacement therapy in postmenopausal women is a reduction of the risk of coronary heart disease.¹ A decrease in low-density lipoproteins and an increase in high-density lipoproteins may be an important mechanism by which estrogen reduces coronary heart disease risk.² Recently, the effect of 17ß-estradiol on endothelium-dependent and endothelium-independent coronary arterial relaxation has been demonstrated.^{3 4 5 6} Modulation of coronary arterial tone could therefore be another mechanism by which estrogen protects the myocardium from ischemia.⁷

The fact that postmenopausal women have a lower incidence of coronary heart disease and myocardial infarction than men of a similar age has led to the hypothesis that testosterone may predispose to coronary artery disease. However, there has been no direct evidence linking testosterone administration to an increased incidence of coronary heart disease and myocardial infarction.

The effect of testosterone on the coronary circulation is unknown; however, there have been reports suggesting that testosterone therapy in men has a beneficial effect on angina pectoris^{8 9 10 11} and on exercise-induced ST segment depression in patients with angina pectoris.¹² A double-blind study was carried out in 50 men who had ST segment depression after exercise.¹² It was shown that after 4 to 8 weeks of treatment with testosterone or placebo, there was a significant decrease in the exercise-induced extent of ST segment depression in patients treated with testosterone. The mechanisms by which testosterone decreased after exercise ST segment depression were not established. The direct effect of testosterone on the coronary artery and any underlying mechanisms of action are not known. The purpose of this study was to assess the effect of testosterone on isolated rabbit coronary arteries and aorta. We examined the possible role of the endothelium, cyclic GMP (cGMP), vasodilator prostanoids, testosterone receptors, potassium conductance, and calcium influx on testosterone-induced coronary and aortic relaxation. We also examined the effect of a number of testosterone analogues on isolated rabbit coronary arteries.

	Methods

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Animals and Tissues
Adult male and nonpregnant female New Zealand White rabbits (weight, 2.5 to 3 kg) were killed by an overdose of pentobarbitone (60 mg/kg) and heparin (150 U/kg), in accordance with our institutional guidelines. The hearts were rapidly removed, and the coronary arteries and aorta were dissected free of connective tissue. Arterial rings were prepared, and, in alternate rings, the endothelium was removed by gentle rubbing with a wooden probe. Each ring was suspended horizontally between two stainless steel parallel hooks for the measurement of isometric tension in individual organ baths containing Krebs solution composed of (mol/L) NaCl 118.3, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, K₂PO₄ 1.2, and glucose 11.1, bubbled with 95% O₂ and 5% CO₂. Temperature was maintained at 37°C. Isometric tension generated by the vascular smooth muscle was measured using a displacement transducer (model UFI, Devices) and recorded on a pen-chart recorder, as previously described.⁵

Relaxing Effect of Testosterone on Precontracted Coronary Arteries and Aorta
Coronary arteries and aortic rings with or without endothelium were stabilized under 1 g of resting tension for 90 minutes before being contracted with prostaglandin F₂ (PGF₂, 3 µmol/L). Testosterone (dissolved in ethanol, 0.1, 1, and 10 µmol/L for coronary rings and 10 and 100 µmol/L for aortic rings) or equivalent ethanol solvent (1 in 1000) was added 7 minutes after the addition of constrictor agent. In one group of experiments, the relaxing effects of testosterone (1 and 10 µmol/L) were compared in rings contracted by PGF₂ (3 µmol/L) or KCl (30 mmol/L).

Effect of N-Nitro-L-Arginine Methyl Ester and Indomethacin on Testosterone-Induced Relaxation
N-nitro-L-arginine methyl ester (L-NAME) is an inhibitor of endothelium-derived relaxing factor (EDRF) synthesis from L-arginine in vascular endothelial cells.¹³ L-NAME (100 µmol/L) was added to coronary arterial and aortic rings with endothelium 20 minutes before being contracted with PGF₂ (3 µmol/L). Indomethacin, an inhibitor of prostanoid synthesis, was dissolved by sonication in an Na₂CO₃ solution. Indomethacin (10 µmol/L) was incubated with endothelium-intact rings for 20 minutes before being precontracted with PGF₂. Testosterone (1, 10, and 100 µmol/L) was subsequently added 7 minutes after the addition of the constrictor agent.

Effect of Methylene Blue on Testosterone-Induced Relaxation
To determine the possible involvement of cGMP in the relaxation induced by testosterone, coronary arterial and aortic rings without endothelium were incubated with methylene blue¹⁴ (10 µmol/L) for 20 minutes before being contracted with PGF₂ (3 µmol/L). Testosterone (1, 10, and 100 µmol/L) was subsequently added.

Effect of Glibenclamide and Barium Chloride on Testosterone-Induced Relaxation
To examine the possible role of ATP-sensitive potassium channels and potassium conductance on testosterone-induced coronary relaxation, glibenclamide (3 µmol/L), an inhibitor of ATP-sensitive potassium channels,¹⁵ or barium chloride (3 mmol/L), a nonspecific inhibitor of potassium channels,¹⁶ was added to coronary artery rings without endothelium 20 minutes before being contracted with PGF₂ (3 µmol/L). The relaxation to testosterone (1, 10, and 100 µmol/L) or equivalent concentrations of ethanol solvent was measured.

Effect of Aminoglutethimide and Flutamide on Testosterone-Induced Relaxation
Aromatase is a tissue enzyme that converts testosterone to estrogen.¹⁷ To examine the possible role of aromatase and testosterone receptors on testosterone-induced coronary relaxation, aminoglutethimide (50 µmol/L), an inhibitor of aromatase, or flutamide (10 µmol/L), a testosterone receptor antagonist,¹⁸ was added to endothelium-denuded coronary artery rings 20 minutes before contraction with PGF₂ (3 µmol/L). The relaxation response to testosterone (1 and 10 µmol/L) then was measured.

Effect of Testosterone and Verapamil on Calcium Concentration–Dependent Contractile Responses in Rabbit Coronary Arteries
Rabbit coronary arterial rings without endothelium were incubated in calcium-free solution containing 0.5 mmol/L EGTA for 10 minutes. Afterward, calcium concentration–dependent contraction curves were performed in K⁺ depolarization medium (K⁺=100 mmol/L). Rings were readjusted in modified Krebs for 20 minutes before being incubated in calcium-free solution containing EGTA (0.5 mmol/L) for a further 10 minutes. Subsequently, rings were incubated with testosterone (1 and 10 µmol/L) or verapamil (1 and 10 µmol/L) or the same concentration of ethanol solvent for 30 minutes. The calcium concentration–dependent contraction curves then were repeated.

Effect of Testosterone Analogues on Precontracted Coronary Arteries
Coronary artery rings were stabilized under 1 g of resting tension for 90 minutes before being contracted with PGF₂ (3 µmmol/L). Testosterone analogues etiocholan-3ß-ol-17-one, epiandrosterone, 17ß-hydroxy-5-androst-1-en-3-one, androst-16-en-3-ol, and testosterone enanthanate (dissolved in ethanol, 1 and 10 µmol/L) were added 7 minutes after the addition of constrictor agent.

Drugs
The following drugs were used: testosterone (4-androsten-17ß-ol-3-one), etiocholan-3ß-ol-17-one (5ß-androstan-3ß-ol-17-one), epiandrosterone (3ß-hydroxy-17-androstanone), 17ß-hydroxy-5-androst-1-en-3-one, androst-16-en-3-ol (3-hydroxy-5-androst-16-ene), testosterone enanthanate (17-[(1-oxoheptyl)oxy]-androst-4-en-3-one), L-NAME, indomethacin, methylene blue, barium chloride, PGF₂, pentobarbitone, aminoglutethimide, flutamide (all supplied by Sigma), and glibenclamide (a gift from Hoechst). All drugs were Analar grade.

Data Analysis
All results are expressed as mean±SEM. Relaxation is expressed as percentage relaxation of contraction induced by PGF₂ (3 µmol/L) or KCl (30 mmol/L). The results were analyzed with a Student's t test for paired and unpaired observations. Each group was compared with the time-matched ethanol solvent control. A probability level of less than .05 was considered significant. For the analogue data, an ANOVA with repeated measurements was used. If a significant F value was found, Scheffé's test for multiple comparisons was used to identify differences among groups. n indicates the number of animals.

	Results

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Relaxing Effect of Testosterone on Precontracted Coronary Arteries and Aorta
PGF₂ (3 µmol/L) and KCl (30 mmol/L) induced comparable contractile responses in coronary arterial rings with and without endothelium (0.7±0.1, 0.7±0.1 g and 0.7±0.05, 0.7±0.05 g, respectively, P>.05). Likewise, PGF₂ (3 µmol/L) induced comparable contractile responses in rabbit aortic rings with and without endothelium (1.12±0.1 and 1.13±0.05 g, respectively). Testosterone (0.1 µmol/L) had no effect on PGF₂-precontracted (3 µmol/L) rabbit coronary arterial rings (Fig 1). However, 1 and 10 µmol/L of testosterone induced significant concentration-related relaxation of contracted rings with or without endothelium from male or female rabbits (compared with time-matched ethanol solvent controls, all P<.01, Fig 1). There were no differences between the relaxation of arteries from male and female rabbits to testosterone 1 and 10 µmol/L (23±5, 74±6 versus 25±5, 71±6, all P>.05, n=8). There were no differences between groups with and without endothelium (P>.05, Fig 1).

View larger version (22K): [in this window] [in a new window]   Figure 1. Bar graph shows the relaxing effect of testosterone (0.1, 1, and 10 µmol/L) on rabbit coronary artery rings with and without endothelium (open and hatched columns, respectively) precontracted with prostaglandin F2 (PGF2) (3 µmol/L). Data are expressed as percentage relaxation of contraction induced by PGF2 (mean±SEM, n=8 in each group). Control indicates time-matched ethanol solvent controls. *Significant differences in comparison with control, P<.01.

Testosterone (1 µmol/L) had no effect on PGF₂- precontracted (3 µmol/L) rabbit aortic rings. However, 10 and 100 µmol/L of testosterone induced significant concentration-related relaxation of contracted rings with and without endothelium from male or female rabbits (compared with time-matched ethanol solvent controls, all P<.01, Fig 2). There were no differences between coronary arteries from male and female rabbits (10±3, 40±3 versus 10±1, 31±7, all P>.05, n=8). There were no differences between groups with and without endothelium (P>.05, Fig 2).

View larger version (18K): [in this window] [in a new window]   Figure 2. Bar graph shows the relaxing effect of testosterone (10 and 100 µmol/L) on rabbit aortic rings with and without endothelium (open and hatched columns, respectively) precontracted with prostaglandin F2 (PGF2) (3 µmol/L). Data are expressed as percentage relaxation of contraction induced by PGF2 (mean±SEM, n=8 in each group). Control indicates time-matched ethanol solvent controls. *Significant differences in comparison with control, P<.01.

Testosterone (1 and 10 µmol/L) induced significantly greater relaxation in rabbit coronary arterial rings than in rabbit aortic rings (P<.05). Testosterone (1 and 10 µmol/L) induced significantly greater relaxation in coronary arterial rings precontracted with PGF₂ (3 µmol/L) than rings precontracted with KCl (30 mmol/L) (P<.01, Fig 3). Representative traces are shown in Fig 4.

View larger version (16K): [in this window] [in a new window]   Figure 3. Bar graph shows testosterone-induced relaxation (1 and 10 µmol/L) in endothelium-denuded rabbit coronary arteries precontracted with prostaglandin F2 (PGF2) (3 µmol/L, open columns) and KCl (30 mmol/L, hatched columns). Data are expressed as percentage relaxation of contraction induced by PGF2 and KCl (mean±SEM, n=8 in each group). *Significant differences in comparison with relaxation induced by testosterone in PGF2-contracted rings, P<.01.

View larger version (8K): [in this window] [in a new window]   Figure 4. Trace of prostaglandin F2 (PGF2)-precontracted (3 µmol/L) and KCl-precontracted (30 mmol/L) rabbit coronary artery preparations showing relaxation to testosterone (TE, 1 and 10 µmol/L).

Effect of N-Nitro-L-Arginine Methyl Ester and Indomethacin on Testosterone-Induced Relaxation
Incubation with L-NAME (100 µmol/L) did not inhibit relaxation induced by testosterone (1 and 10 µmol/L) in either rabbit coronary arterial rings (14±2 versus 23±5 and 55±8 versus 74±6, respectively, P>.05) or rabbit aortic rings (12±1 versus 12±5 and 37±3 versus 32±6, respectively, P>.05) with endothelium. Incubation with the prostaglandin synthetase inhibitor indomethacin (10 µmol/L) did not affect the relaxation induced by testosterone (1 and 10 µmol/L) in rabbit coronary arterial rings (14±2 versus 23±5 and 62±9 versus 74±6, respectively, P>.05) or aortic rings (11±1 versus 12±5 and 40±3 versus 32±6, respectively, P>.05) with endothelium.

Effect of Methylene Blue on Testosterone-Induced Relaxation
Incubation with methylene blue (10 µmol/L) for 20 minutes before contraction with PGF₂ (3 µmol/L) had no effect on relaxation induced by testosterone (1 and 10 µmol/L) in rabbit coronary arterial rings (15±3 versus 23±5 and 68±7 versus 74±6, respectively, P>.05) or testosterone (10 and 100 µmol/L) in aortic rings (11±3 versus 12±5 and 30±7 versus 32±6, respectively, P>.05) without endothelium.

Effect of Glibenclamide and Barium Chloride on Testosterone-Induced Relaxation
Glibenclamide (3 µmol/L) did not affect the relaxing effect of testosterone. Barium chloride (3 mmol/L) partially but significantly reduced testosterone-induced relaxation in rabbit coronary arterial rings without endothelium (n=8, P<.05, Fig 5).

View larger version (23K): [in this window] [in a new window]   Figure 5. Bar graph shows the effect of barium chloride (3 mmol/L, wide-hatched columns) and glibenclamide (3 µmol/L, narrow-hatched columns) on relaxation induced by testosterone (1 and 10 µmol/L, open columns) in endothelium-denuded rabbit coronary arteries precontracted with prostaglandin F2 (PGF2) (3 µmol/L). Data are expressed as percentage of contraction induced by PGF2 (mean±SEM, n=6 in each group). *Significant differences in comparison with relaxation induced by testosterone, P<.05.

Effect of Aminoglutethimide and Flutamide on Testosterone-Induced Relaxation
Aminoglutethimide (50 µmol/L) did not affect the relaxing effect of testosterone (10 µmol/L) in coronary arterial rings (68±7% and 70±7% before and after aminoglutethimide, respectively, n=6, P>.05). Flutamide (10 µmol/L) did not influence testosterone-induced (1 and 10 µmol/L) relaxation in coronary arterial rings without endothelium (17±3, 72±7 and 16±3, 71±3 before and after flutamide, respectively, n=6, P>.05).

Effect of Testosterone and Verapamil on Calcium Concentration–Dependent Contractile Responses in Rabbit Coronary Arteries
The calcium concentration–dependent contraction curves in K⁺ depolarization medium were not affected by testosterone (1 and 10 µmol/L) in rabbit coronary arterial rings without endothelium. The -log EC₅₀s of calcium in control rings and after incubation with testosterone (1 and 10 µmol/L) for 30 minutes were 3.8±0.2, 3.5±0.2, and 3.6±0.15, respectively (Fig 6). In contrast, the concentration-dependent contraction curves in K⁺ depolarization medium were shifted to the right in a dose-dependent manner after incubation with verapamil (1 and 10 µmol/L) in rabbit coronary arterial rings without endothelium. Maximal contraction was reduced to 52±9% and 18±5%, respectively. The -log EC₅₀s of calcium in control and after incubation with verapamil (1 and 10 µmol/L) for 30 minutes were 3.7±0.1, 2.8±0.18, and 1.8±0.1, respectively (Fig 7).

View larger version (13K): [in this window] [in a new window]   Figure 6. Line plot shows the effect of testosterone (1 and 10 µmol/L) on calcium concentration–dependent contraction curves in rabbit coronary arteries without endothelium. Indicates control; , 1 µmol/L testosterone; and , 10 µmol/L testosterone. Data are expressed as percentage of maximal contraction induced by calcium in controls (mean±SEM, n=8 in each group).

View larger version (14K): [in this window] [in a new window]   Figure 7. Line plot shows the effect of verapamil (1 and 10 µmol/L) on calcium concentration–dependent contraction curves in rabbit coronary arteries without endothelium. Indicates control; , 1 µmol/L verapamil; and , 10 µmol/L verapamil. Data are expressed as percentage of maximal contraction induced by calcium in controls (mean±SEM, n=8 in each group).

Effect of Testosterone Analogues on Precontracted Coronary Arteries
There were significant differences in the relaxing potency of testosterone analogues (1 and 10 µmol/L) on precontracted rabbit coronary arterial rings (Fig 8). There were no significant differences between the relaxation induced by testosterone (1 and 10 µmol/L), etiocholan-3ß-ol-17-one, and epiandrosterone; however, there were significant differences between testosterone-induced relaxation and that induced by 17ß-hydroxy-5-androst-1-en-3-one, androst-16-en-3-ol, and testosterone enanthanate.

View larger version (27K): [in this window] [in a new window]   Figure 8. Bar graph shows the effect of testosterone (Te, 1 and 10 µmol/L), etiocholan-3ß-ol-17-one (Eti, 1 and 10 µmol/L), epiandrosterone (Epi, 1 and 10 µmol/L), 17ß-hydroxy-5-androst-1-en-3-one (Hydro, 1 and 10 µmol/L), androst-16-en-3-ol (Andro, 1 and 10 µmol/L), and testosterone enanthanate (Tee, 1 and 10 µmol/L) on rabbit coronary artery rings with and without endothelium. Significant differences in comparison with testosterone-induced relaxation: *P<.05, **P<.01, and ***P<.001 (ANOVA).

	Discussion

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We have demonstrated that testosterone (1, 10, and 100 µmol/L) induces relaxation in precontracted rabbit coronary arteries and aorta with or without endothelium. No difference was seen between arteries from male and female rabbits. Barium chloride partially inhibited the relaxation, and the relaxing response to testosterone was greater in PGF₂-contracted rings compared with KCl-contracted rings. Neither L-NAME, methylene blue, indomethacin, animoglutethimide, flutamide, nor glibenclamide affected this relaxation. Testosterone did not shift the calcium concentration–dependent contraction curve in high K⁺ solution (100 mmol/L) in rabbit coronary artery preparations.

Early studies from the 1940s assessed the effect of intramuscular testosterone, usually 25 mg given 2 to 5 times per week, on a variety of clinical parameters including anginal symptoms and crude ECG assessments.^{8 9 10} These studies suffer from the fact that the numbers of patients studied were small (7, 9, and 20, respectively), there was no documentation of coronary artery disease, and very few patients received placebo. A later study by Lesser¹¹ reported marked clinical improvement of angina pectoris in 91 out of 100 patients treated with 15 to 25 intramuscular injections of 25 mg of testosterone propionate given over periods varying from several months to 5 years. Again, this study provided no objective evidence of a benefit in myocardial ischemia and no documentation of coronary artery disease. In this study, 5 patients were given six consecutive injections of sesame oil as control, and none of these patients showed any change in symptoms. One of the commonly cited theoretical mechanisms of the beneficial effect of testosterone on anginal symptoms in these studies was coronary vasodilatation.^{8 10 11} In a later study, testosterone treatment for several weeks reduced objective evidence of exercise-induced myocardial ischemia in patients with angina pectoris.¹² Possible mechanisms discussed for this beneficial effect included an improvement of oxygen-carrying capacity of red cells¹⁹ and an increase in blood hemoglobin levels.²⁰ Dilatation of the coronary arteries or their collaterals also was suggested to account for the beneficial effect; however, this hypothesis has never been tested. Testosterone is highly protein bound in plasma²¹ ; 98% of testosterone is bound to sex hormone–binding globulin, albumin, and other proteins. This may result in substantially different concentrations at different sites, including the smooth muscle of arteries, in vivo. Physiological effects may therefore occur despite lower plasma concentrations of the hormone.

Some of the increased risk for coronary heart disease in men has been attributed to differences in lipoprotein levels; in particular, high-density lipoprotein cholesterol (HDL-C), a protective factor, is higher in women. There are some reports that testosterone substitution in men is associated with decreased serum HDL-C levels.^{22 23} However, the data are by no means consistent, since testosterone replacement in elderly men caused a decrease in total cholesterol without a change in HDL-C cholesterol,²⁴ and testosterone treatment may have other beneficial effects on cardiovascular and diabetic risk factors such as insulin resistance.²⁵ The effect of physiological levels of testosterone in the control of lipoproteins and cardiovascular risk also is not clear. Some studies suggest a suppressive effect on HDL-C,²⁶ whereas other studies suggest that testosterone levels are favorably associated with cardiovascular risk.^{27 28} It also has been shown that hypotestosteronemia is associated with an adverse cardiovascular risk^{29 30} and that testosterone replacement in such men reverses this adverse risk.³¹ The data on the unfavorable effect of testosterone replacement on cardiovascular risk are therefore not established or clear.

Acetylcholine induces endothelium-dependent vascular relaxation mediated by the release of EDRF.³² We have demonstrated that testosterone induced an equal degree of relaxation in rabbit coronary arteries and aorta with and without endothelium. L-NAME, an inhibitor of EDRF synthesis,¹³ did not affect the relaxation by testosterone. Methylene blue, an inhibitor of EDRF- induced increase of cGMP,¹⁴ also had no effect on relaxation induced by testosterone. Our results suggest that the in vitro acute relaxation of rabbit coronary arteries and aorta by testosterone is independent of EDRF.

Indomethacin inhibits the synthesis of prostaglandins.^{33 34} Indomethacin markedly inhibits the transient relaxation induced by arachidonic acid in rabbit coronary arteries.⁴ However, indomethacin did not affect testosterone-induced relaxation in endothelium-intact coronary arteries. These results indicate that the release of vasodilator prostanoids is not involved in testosterone-induced coronary relaxation in vitro.

Glibenclamide, an inhibitor of ATP-sensitive potassium channels,¹⁵ did not affect testosterone-induced relaxation. Barium chloride, a nonspecific inhibitor of potassium channels,¹⁶ did attenuate the relaxing response to testosterone in rabbit coronary arteries, suggesting that alterations of potassium conductance may be involved partially in the mechanism of relaxation. At concentrations less than those used in this study, barium chloride has been shown to reverse the nonendothelium vasorelaxing actions of the potassium channel–opening drugs diazoxide, cromakalim, and pinacidil in norepinephrine-contracted (10 µmol/L) rabbit mesenteric artery rings.¹⁵ Testosterone is much more effective in relaxing PGF₂-contracted tissues than those contracted with high concentrations of potassium. The reason for this difference is not clear, but similar differences have been demonstrated for the relaxing responses to potassium channel openers such as pinacidil.³⁵ This differential effect is indicative of potassium channel opening³⁶ and supports the possibility that the testosterone-induced relaxation may involve potassium channel opening, since the relaxation was partly inhibited by barium chloride.

There is no evidence that testosterone receptors exist in vascular or cardiac tissues. We did assess the effect of the nonsteroidal antiandrogen flutamide¹⁸ on testosterone-induced relaxation. The other potential relaxing mechanism of testosterone is by its conversion to estradiol via the aromatase pathway.¹⁷ Therefore, we investigated the relaxing effect in the presence of aminoglutethimide, which is a competitive nonsteroidal aromatase inhibitor. This substance blocks the conversion of androgenic prohormones to estrogen.¹⁷ Neither aminoglutethimide nor flutamide affected the relaxation responses to testosterone, suggesting that neither the testosterone receptor nor its conversion to estrogen is involved in the mechanism of smooth muscle relaxation.

Potential sensitive calcium channels are activated by depolarization of the plasma membrane when the extracellular K⁺ concentration is increased. Incubation with testosterone did not shift the calcium concentration–dependent contraction curves to the right in high K⁺ depolarization medium in rabbit coronary arteries without endothelium. These results suggest that testosterone does not have a calcium-antagonistic property in these vascular preparations.

The concentrations that induce relaxation of rabbit coronary arterial preparations in vitro (1 and 10 µmol/L) are approximately 50 and 100 times greater than those found in normal male volunteers (21±1 nmol/L) and approximately 10 times greater than those found in New Zealand White rabbits (77±2 to 638±11 nmol/L).³⁷ However, the concentrations of testosterone produced by intramuscular injections of 25 mg of the hormone produce supraphysiological peak blood levels in hypogonadal men.³⁸ These levels are approximately 10 times less than those used in this in vitro study, which induced coronary relaxation. The plasma levels achieved in those normal men treated by Jaffe¹² may have been equivalent to concentrations used in this study. Direct coronary relaxation therefore may have been one of the mechanisms of the beneficial effect of testosterone on angina in those patients as hypothesized in this article.¹² It is well recognized that discrepancies exist between the concentration of agents that induce in vivo changes and those that induce in vitro smooth muscle–relaxing responses in the organ bath. Examples would be calcium channel blockers³⁹ and potassium channel–opening agents.⁴⁰ Cromakalim, a potassium channel opener, has been shown acutely to reduce systolic arterial pressure and systemic vascular resistance in patients with angina pectoris at plasma concentrations of 2 to 3x10^-8 mol/L,⁴¹ whereas relaxing effects of cromakalim in phenylephrine contracted rat aorta in vitro only occurred at 10^-6 and 10^-5 mol/L concentrations.⁴² Likewise, cromakalim-induced relaxation of PGF₂-contracted (10 µmol/L) pig coronary arteries in vitro occurred only at 10^-6 and 10^-5 mol/L. These data would be analogous to the discrepancy between the concentration of testosterone required to induce relaxation of coronary arteries in vitro and those concentrations found in vivo.

The testosterone analogues used in this study were chosen on the basis of previous work demonstrating a nongenomic structure-activity relation of testosterone analogues in a New Zealand White rabbit model of a thyroid hormone–responsive membrane calcium-ATPase.⁴³ This sex steroid–thyroid hormone interaction at or near the calcium-ATPase site in the rabbit reticulocyte is novel in that it is at the cell membrane and represents a previously unrecognized ability of steroids to directly modulate the thyroid hormone. It was demonstrated that 5ß-androstanes were active, whereas 5-androstanes were less active. Within the 5ß-androstanes, activity was dependent on at least one hydroxyl group at the C3 or C17 position. In a similar way, we have demonstrated that the presence of a hydroxyl group at the 17ß position may be important, since those analogues with no hydroxyl group (androst-16-en-3-ol) or with esterification of the 17ß-hydroxyl group (testosterone enanthanate) result in a significant decrease in relaxing potency. It was also found that, compared with testosterone, analogues derived by oxidation, substitution, or deletion at the C3, C17, or C19 positions were less active.⁴³ The reason for this was unclear but does suggest a structure-activity relation. It is interesting to note that the 5ß-configuration results in a marked angulation of the A-ring relative to the plane of the remaining rings of the steroid. The flat steroid conformation made by the all-trans–anti-trans ring junctures would appear to decrease activity, since the 5-androstane analogue (17ß-hydroxy-5-androst-1-en-3-one) resulted in less coronary artery relaxation. These data would suggest that testosterone is interacting with the plasma membrane and may be affecting potassium conductance by an interaction with the potassium channel. The time course of the relaxing response and the inability of the testosterone receptor antagonist to affect the relaxation would also support a nongenomic mechanism of relaxation. The mechanism of such an interaction is undetermined; however, hydrophobic hormone or drug molecules may be orientated selectively in the lipid bilayer to enhance the efficacy of binding of such molecules to cell membrane receptor sites. Such a novel action may account for a nonconformity of such molecules to traditional binding kinetics, as previously postulated.⁴³

Conclusions
We have demonstrated that testosterone induces endothelium-independent relaxation in isolated rabbit coronary artery and aortic preparations. The mechanism may involve, in part, the vascular smooth muscle potassium channel. Testosterone may play a role in the regulation of coronary tone, and this may be one of the explanations as to why testosterone has previously been shown to demonstrate beneficial effects on anginal symptoms and on parameters of myocardial ischemia in patients treated with this hormone. Further work will be required to establish if this vascular effect has any therapeutic potential in patients with coronary heart disease.

	Acknowledgments

 
This study was supported by a grant from the British Heart Foundation.

Received September 8, 1994; accepted September 23, 1994.

	References

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