O. Yildiz, M. Seyrek

Department of Pharmacology, Gulhane School of Medicine, Ankara, Turkey.

Bibiliography: DOI 10.1055/s-2007-949657 Exp Clin Endocrinol Diabetes 2007; 115:1-6 © J.A. Barth Verlag in Georg Thieme Verlag KG • Stuttgart • New York • ISSN 0947-7349

O. Yildiz, Department of Pharmacology • Gulhane School of Medicine • 06018 Ankara • Turkey Tel.: +90/312/3044766 Fax: +90/312/304215 0; [email protected]: [email protected].

Abstract

The increased incidence of cardiovascular disease in man compared with premenopausal women suggests an unfavourable effect of male sex hor-mone testosterone on the cardiovascular system. However, numerous clinical and epidemiological studies reported a controversial relationship between testosterone and cardiovascular disease. Furthermore, an increasing amount of evidence indicate that testosterone can exert acute vasorelaxing effects, via non-genomic mechanisms. These effects involve primarily the vascular smooth muscle, without requiring the presence of endothelium, although an endothelial contribution is apparent in some studies. To date, the mechanism behind the vasodilatory action of testosterone is still under debate and might be through either activation of K⁺ channels or blockade of Ca²⁺ channels in vascular muscle cells. The purpose of this article is to review the evidence regarding the vasodilating effect of testosterone as well as its mechanism of action.

Key words: testosterone, vasodilation, vascular smooth muscle, nongenomic effects.

Introduction

In men, the risk of developing coronary artery disease and hypertension is much higher than in premenopausal women (Messerli et al., 1987; Khaw et al., 1988). However, cardiovascular protection during the reproductive age is lost after menopause, and is recovered in postmenopausal women who receive estrogen replacement therapy (Stampfer et al., 1991). This has led to supposition that estrogens are beneficial and androgens are detrimental to the cardiovascular system.

There is now a plethora of evidence that estrogens may play an important cardiovascular protective role not only by means of long-term systemic effects, like those on serum lipid profile, but also by means of direct effects of vascular structure and function, such as inhibition of vascular growth modulation of vascular tone (Simonchini and Genazzani, 2003; Cho et al., 2003). Of the effects previously studied, the vasodilating effect induced by estrogens has been well-documented. The mechanism of action of estrogens appears to involve genomic and non-genomic mechanisms that cause increase and release of vasodilatory agents such as nitric oxide (NO) and prostacyclin, adenosine, cAMP and cGMP (Belfort et al., 1996; Darkow et al., 1997; Dubey and Jackson, 2001) Potassium- channel opening and calcium antagonistic actions have also been reported (White et al., 1995; Valverde et al., 1999; White et al., 2002; Han et al., 2006).

On the other hand, testosterone has classically been considered to exert an unfavourable effect upon the cardiovascular system. However, numerous clinical and epidemiological studies reported a controversial relationship between testosterone and cardiovascular disease. For example, low circulating testosterone levels in men are positively correlated with numerous risk factors for coronary heart disease (English et al., 1997 and 2000a). Evidence also supports a beneficial effect of testosterone on the blood lipid profile and against atheroma formation (English et al., 1997; Duell et al., 1990; Khaw et al., 1991; Hromadowa et al., 1991a and b; Alexandersen et al., 1999). Furthermore, acute intracoronary or intravenous infusion of testosterone also provides rapid improvements in myocardial ischemia (Rosano et al., 1999; Webb et al., 1999a). Finally, testosterone replacement therapy improves myocardial ischemia in patients with coronary artery disease, an effect presumably due to testosterone -induced coronary vasodilatation (English et al., 2000b). Previous animal studies have dem-onstrated that testosterone acts as a direct coronary vasodilator in a variety of species, including rabbit, dog, pig, and rat, both in vivo (Chou et al., 1996) and in vitro (Yue et al., 1995; Murphy and Khalil, 1999; Crews and Khalil 1999a and b; English et al., 2000c; English et al., 2002). Testosterone is also reported to exhibit a vasodilatory action in thoracic aortae (Yue et al., 1995; Perusquia et al., 1996; Honda et al., 1999; Ding and Stallone, 2001) and in vessels isolated from the mesenteric (Tep-areenan et al., 2002) and pulmonary (English et al., 2001; Jones et al., 2002) vasculature of experimental animals and more recently in isolated human pulmonary and mesenteric arteries (Jones et al., 2003a). We have recently shown that testosterone relaxes human internal mammary (IMA) and radial (RA) but not umbilical arteries (UA) (Yildiz et al., 2005a).

Mechanisms of testosterone-induced vasodilation; Involvement of the androgen receptor (AR)

Numerous experimental studies have been conducted to make clear the mechanisms of testosterone-induced vasodilation. Testosterone is classically known to regulate cellular function via interaction with the nuclear AR. In the bloodstream, most of circulating testosterone is bound to serum proteins, mainly sex hormone binding protein (SHBG) and albumin. In target cells testosterone can directly bind to the nuclear AR, or it is converted to dihydrotestosterone, which binds in turn to the AR. The hormone-receptor complex directly interacts with the nuclear DNA, modulating the transcription of androgen responsive genes. This process of protein synthesis usually takes at least 40 min to hours.

In trying to explain the vasodilatory mechanism of action of testosterone, the initial studies focused on the degree of involvement these classical steroid transcription mediated pathways played in the response (Yue et al., 1995; Chou et al., 1996; Ding and Stallone, 2001; Tep-areenan et al., 2002; Jones et al., 2002). In this respect, it has been shown that the latency for the vasodilating effect of testosterone is fast and reversible. It seems unlikely that testosterone-mediated vasodilatation would involve classical AR and genomic signalling pathway, because the response is evident within minutes of application and is maximal by 20 min, not hours. Moreover, additional substantial evidence is provided by findings that testosterone-induced dilatation is not attenuated either by pre-treatment with the AR blocker flutamide (Yue et al., 1995; Chou et al., 1996; Ding and Stallone, 2001; Tep-areenan et al., 2002; Jones et al., 2002), or by the covalent linkage of testosterone analogues to albumin, thus preventing endocytosis into the smooth muscle cell (Ding and Stallone, 2001; English et al., 2000c). In addition, blockers of transcription (e.g. actinomycin D) and translation (e.g. cyclohex- imide) does not inhibit testosterone-induced vasodilation (Teoh et al., 2000). Furthermore, it has also been demonstrated that testosterone-mediated vasodilatation is maintained in vessels isolated from testicular feminized mice, which lack a functional AR (Jones et al., 2003b). Previously, it was proposed that the relative polarity of the testosterone molecule determined its own vasorelaxation efficacy (Ding and Stallone, 2001). In our laboratory, we have tested the efficacy of various testosterone analogs in human IMA and RA (Yildiz et al., 2005a and unpublished data). Testosterone propionate is the most polar, least lipid soluble and smallest molecular weight (MW: 344.5) among the other esterified testosterone analogs we have tested. Our data support the suggestion that testosterone-induced vasorelaxation is a non-genomic and structurally specific effect of the testosterone molecule, which is enhanced in more polar analogs that have a lower permeability to the vascular smooth muscle cell membrane (Ding and Stallone, 2001).

Involvement of aromatization

Potential conversion of testosterone to estrogen is another important concept to be addressed. In this respect, previous studies have demonstrated that inhibition of aromatase activity or estrogen receptor antagonism had no effect of testosterone- induced vasodilation (Yue et al., 1995; Tep-areenan et al., 2002). Moreover, similar vasodilatation to testosterone was triggered by non-aromatizable dihydrotestosterone (Deenadayalu et al., 2001). In our laboratory, we used tamoxifen, an estrogen receptor antagonist and protein kinase C inhibitor, and letrozole, a non-steroidal aromatase inhibitor, to test the role of aromatisation in testosterone-induced relaxation in IMA.

While tamoxifen inhibited testosterone- induced relaxation, letrozole had no effect (Gul et al., 2004).

This suggested that aromatisation did not occur in human IMA, thus estrogen did not contribute on testosterone-induced relaxation.

Involvement of endogenous vasodilators

A number of studies have investigated the involvement of dilator prostanoids and endothelium derived nitric oxide (Yue et al., 1995; Chou et al., 1996; Costarella et al., 1996; Honda et al., 1999; Ding and Stallone, 2001; Tepareenan et al., 2002; Jones et al., 2002; Tep-areenan et al., 2003). However, some of these studies report that indomethacin, an inhibitor of cyclo-oxygenase responsible for the synthesis of the cyclic endoperoxide from arachidonate, has no inhibitory effect upon testosterone mediated relaxation (Chou et al., 1996; Yue et al., 1995; Honda et al., Jones et al., 2002). Similarly, testosterone-induced vasodilatation has been reported to be preserved in endothelial denuded vessels (Yue et al., 1995; Murphy and Khalil, 1999; Crews and Khalil 1999a and b; Deenadayalu et al., 2001; Perusquia et al., 1996; Honda et al., 1999; Hishikawa et al., 1995) or in the presence of inhibitors of nitric oxide syntase or guanylate cylase (Yue et al., 1995; Deenadayalu et al., 2001; Honda et al., 1999; Jones et al., 2002). In addition, previous studies also showed that testosterone had no effects on NOS activity in human and bovine aortic endothelial cells (Hishikawa et al., 1995; Goetz et al., 1999). In canine coronary artery (English et al., 1997) and rat mesenteric arterial bed (Tepareenan et al., 2002) testosterone causes acute endothelium-dependent relaxation, mediated by nitric oxide. However, nitric oxide synthase (NOS) inhibition has no effect on relaxations to testosterone in rabbit coronary artery (Yue et al., 1995), rat aorta (Honda et al., 1999) and pulmonary artery (Jones et al., 2002). In addition, other studies with canine, rabbit, and pig coronary arteries (Yue et al., 1995; Chou et al., 1996; Deenadayalu et al., 2001) and rat thoracic aorta and mesenteric arterial bed (Honda et al., 1999; Ding and Stallone et al., 2001; Tep- areenan et al., 2002) have reported that endothelium and NO - independent vasorelaxations induced by testosterone. We have recently shown that testosterone -induced vasodilatation in human internal mammary and radial arteries is preserved in endothelial denuded vessels or in the presence of inhibitors of nitric oxide syntase or guanylate cylase (Yildiz et al., 2005a and unpublished observation).

Potassium - channel opening and calcium antagonistic actions

A number of studies have provided evidence of a modulatory role for testosterone upon potassium channel function. Chou et al. (1996) and Honda et al. (1999) previously showed that glibenclamide, an ATP-sensitive K⁺ channel (K_ATP) inhibitor, reduced vasorelaxation to testosterone in dog coronary artery and rat thoracic aorta, respectively. On the other hand, Ding and Stallone (2001) demonstrated that neither incubation with glibenclamide nor tetraetylammonium (TEA), a non-selective K⁺ channel inhibitor, had any effect on the vasodilatory response to testosterone in rat thoracic aorta. But they showed that incubation with 4-aminopyridine (4-AP), a voltage-gated K⁺ channel (K_v) inhibitor, significantly reduced vasorelaxation to testosterone and they therefore proposed that testosterone acts via K_v channel opening action. Deenadayalu et al. (2001) recently demonstrated that TEA inhibited vasorelaxation to testosterone in pig coronary arteries. Moreover, Deenadayalu et al. (2001) confirmed that testosterone opened large-conductance Ca²⁺-activated K⁺ channels (BK_Ca) in single pig coronary myocytes by direct evidence from patch-clamp studies. In our recent study, we have also observed an inhibitory effect of TEA, but not glibenclamide and 4-AP, on vasorelaxation to testosterone, suggesting that testosterone -mediated vasodilatation may occur via activation of BK_Ca channels in human internal mammary artery (Yildiz et al., 2005a). However in human radial artery, glibenclamide, but no other K⁺ channel inhibitors, reduced vasorelaxation to testosterone, suggesting involvement of K_ATP channels (unpublished observations).

Interestingly, Deenadayalu et al. (2001) reported a decrease in the response to testosterone under similar conditions with our study in IMA (Yildiz et al., 2005a), namely less testosterone relaxation after KCl-precontraction compared to that after PGF₂α-precontraction. In our study, we have found a significant reduction in the efficacy of testosterone in vessels precontracted with KCl compared to PGF₂α. An explanation of these results may lie with the potassium channel function which is compromised in conjunction with the vasodilatory efficacy of testosterone. An alternative explanation of the dilatory action testosterone may lie with the types of calcium channels involved. PGF₂α acts primarily at the prostanoid receptors gated directly to receptor- operated calcium channels (ROCCs) (Tosun et al., 1997 ). This receptor stimulation also results in phosphatidylinositol 4,5 bisphosphate hydrolysis (Raymond et al., 1983) with resultant generation of inositol triphospate, which triggers calcium release from the intracellular stores (Berridge, 1987). The high extracellular potassium gradient generated by milimolar KCl triggers membrane depolarization with subsequent activation of voltage-operated calcium channels (VOCCs). Recently, Jones et al. (Jones et al., 2003c and 2004) reviewed the literature and proposed that the variance in the testosterone -induced dilatation following preconconstiction with KCl and PGF₂α, could be due to a differing inhibitory efficiacy of testosterone upon ROCCs and VOCCs.

A number of previous studies in isolated vessel preparations have supported the idea that testosterone acts as a Ca²⁺ channel antagonist, by inhibiting VOCCs. It has previously been demon-strated that testosterone inhibits VOCCs in isolated rat pulmonary (Jones et al., 2002), rat coronary (English et al., 2002) and porcine coronary arteries (Crews and Khalil, 1999b). These studies have indicated that of the multiple pathways underlying Ca²⁺ signalling and homeostasis in vascular smooth muscle, testosterone appears specifically to target voltage-gated Ca²⁺ entry via L-type Ca²⁺ channels, whilst agonist mobilization of Ca²⁺ and consequent capacitative calcium entry remain unaffected (Jones et al., 2002; English et al., 2002; Crews and Khalil, 1999b; Scragg et al., 2004). Moreover, Scragg et al. (2004) have employed whole-cell patch clamp recordings under non-physiological conditions and they have shown that testosterone inhibits L- type Ca²⁺ channels. In recent study, Hall et al. (2006) have employed fluorometric recordings and they have provided evidence that testosterone can block both native and recombinant L-type voltage-gated Ca²⁺ channels with IC₅₀ values within or close to levels of circulating testosterone concentrations. VOCC inhibition by testosterone is rapid (within 2 minutes), making it unlikely to be mediated through a genomic effect. Hall et al. (2006) have also observed that nifedipine, a L-type Ca²⁺ channel blocker, blocks the effects of testosterone and they have proposed that some of the beneficial cardiovascular effects of testosterone arise from its ability to act like a dihydropyridine antihypertensive to suppress Ca²⁺ entry and so promote vasodi-lation. In our laboratory, we have observed that testosterone at concentrations of 100μM and higher inhibited CaCl₂ -induced contractions in human IMA (Yildiz et al., 2005a). Consistent with our observation, it has been recently demonstrated that testo-sterone, at high concentrations, inhibits Ca²⁺ influx in rat aorta (Tep-areenan et al., 2003).

To date, the mechanism behind the vasodilatory action of testo-sterone is still under debate and might be through either activation of K⁺ channels or direct blockade of Ca²⁺ channels in vascular muscle cells. Deenadayalu et al. (2001) convincingly demonstrated that testosterone-mediated vasodilatation occurs via activation of K⁺ channels by direct evidence from patch- clamp study. On the other hand, only the study of Hall et al. (2006) has provided evidence for a direct calcium antagonistic effect of testosterone, so far. Indeed, either mechanism would allow vasodilatation from reduced Ca²⁺ influx through VOCCs (S Fig.1).

Figure 1. Proposed model for actions of testosterone. The opening of K⁺ channels in the cell membrane of smooth muscle cells in arteries by testosterone increases K⁺ efflux, which causes membrane potential hyperpolarization. This closes voltage-operated calcium channels (VOCCs), decreasing Ca²⁺ entry, which leads to vasodilation. K_ATP: ATP-sensitive K⁺ channel, BK_Ca: large-conductance Ca²⁺ -activated K⁺ channel, K_v: a voltage-gated K⁺ channel.

Contradictory effects of testosterone on potassium channels

There is a complex-mixture of vessel-type dependent mechanisms reported in previous studies about the type of the K⁺ channel involved in the vasodilatory action of testosterone. In rabbit, dog and pig coronary conduit arteries, testosterone acts via BK_Ca and K_v channel opening action, however testosterone - induced vasodilation occurs via K_ATP channel opening action in dog coronary resistance arteries (Yue et al., 1995; Chou et al., 1996; Deenadayalu et al., 2001). Accordingly, testosterone acts via BK_Ca channel opening action in an human conduit artery, the IMA (Yildiz et al., 2005a). However, RA shows a resistance vessel characteristics as elastomuscular wall structure and a small diameter, and testosterone - induced vasodilation occurs via K_ATP channel opening action in RA similar to resistance arteries (unpublished observations). Therefore, it may be suggested that testosterone preferentially stimulates the K_v and K_Ca channels in large conductance vessels, and the K_ATP channel in small resistance vessels. In fact, there are some differences in the vascular reactivity between large conductance and small resistance arteries Yokoshiki and Sperelakis, 2003. For example, it was reported that adenosine, a metabolic regulator of coronary blood flow (probably via activating K_ATP channels and inhibiting L-type Ca²⁺ current (t), preferentially blocked the action potential in the small coronary artery, but had no effect on the action potential in the large coronary artery (Harder et al., 1979). Likewise, the coronary dilating actions levcromakalim, of a K_ATP channel opening drug, were heterogeneous, and it was more sensitive in small resistant coronary arteries (Sato et al., 1994). It was also reported that the resting tone of large coronary conductance vessels was little affected by glibenclamide (Shimizu et al., 2000), whereas that of small resistance vessels was affected. Hence, the tone of the small vessels is likely to be regulated by the glibenclamidesensitive K_ATP channel (Yokoshiki et al., 1997). Another important finding is that testosterone relaxes human internal mammary and radial arteries but it has no vasodilatory effect on human umbilical arteries (HUA) (Yildiz et al., 2005a). HUA is unique, since it shows a conductance vessel characteristics as elastic wall structure and it has a small diameter. Functional K_v and BK_Ca ca channels are present in HUA (Yildiz et al., 2006a). However, this artery seems to lack the non-genomic signalling pathway involved in testosterone-induced vasodilation.

Potential factors influencing testosterone responses

An important point of the previous studies which should be addressed was that high concentrations of testosterone (in the micromolar range) were required to elicit vasodilation. Most of these studies were carried out in isolated vessel preparations, therefore the effects of testosterone might occur only at supra- physiological concentrations. There may be a concentration gradient from the perfusate down to the cell membrane as a result of impaired delivery of the testosterone molecule or binding to various proteins. There may be a consequence of access to smooth muscle ion channels; this could be restricted in more intact preparations lacking their natural blood supply as the lipophilic androgen would need to cross some barriers before reaching its presumed target on smooth muscle cells (Hall et al. 2006).

Another point of the previous studies which should be looked up was the latency for the vasodilating effect of testosterone. In most of the mechanistic studies cited in this review, free testosterone analogues were used, and the vasodilatory action of testosterone was rapid, occuring within minutes. As mentioned above, the covalent linkage of testosterone analogues to a macromolecule, such as albumin, has no effect upon the timescale and magnitude of the vasodilatory response to testosterone (English et al., 2000d; Ding and Stallone, 2001). Therefore, the conjugation of testosterone to albumin eliminates the permeability of testosterone to the cell membrane, and thereby, these findings again suggest a non-genomic action.

The issue of how testosterone is solved and delivered is of great importance. Different studies in humans and animals have used different solvents in different concentrations (e.g. ethanol and dimethysulphoxide). Solvents have unselective effects on ion channel activity (Kunz et al., 2006), within miliseconds, which could interfere with selective actions of testosterone. In previous studies however, no significant relaxant effect of final con-centrations of the solvents has been reported (Yue et al., 1995; Chou et al., 1996; Costarella et al., 1996; Honda et al., 1999; English et al., 2000d , Ding and Stallone, 2001; Tep-areenan et al., 2002; Jones et al., 2002; Tep-areenan et al., 2003). In our experiments, we did not let the final ethanol concentration in the bath to exceed 0.1 % (v/v), since it might induce relaxation itself (Yildiz et al., 2005a).

Variability of testosterone -induced vasodilatory responses

An interesting finding in our observations was the marked variability of responses to testosterone in various subjects in IMA (Yildiz et al., 2005a). We have recently investigated the relationship of this variability with cardiovascular risk factors (Yildiz et al., 2005b). Cumulative relaxations to testosterone after precontraction with KCl have been examined in IMA segments from patients with identified cardiovascular risk factors such as hypercholesterolemia, diabetes, hypertension, smoking, age, gender, body mass index (BMI), and number of occluded vessels. Testosterone responses were significantly diminished in subjects with 3 compared with 1 risk factor. Hypercholesterolemia has independently influenced testosterone responses by significantly decreasing its maximum, and smoking has significantly decreased the sensitivity to testosterone. In our study, aging is one of the factors related to diminished testosterone responses. In accordance, English et al. (2000b) have also demonstrated that aging reduces the testosterone -induced relaxation in coronary arteries from male Wistar rats in vitro. We have concluded that the variability observed in testosterone-induced vascular relaxations may in part be related to differences in risk factors present among the individuals studied (Yildiz et al., 2005b).

Clinical relevance and future research

Testosterone-induced vasodilation might protect against cardio-vascular disease, possibly by arterial vasodilation and reduced blood pressure (Thompson and Khalil, 1999; Wu and von Eck- ardstein, 2003; Littleton-Kearney and Hurn, 2004); thus animal studies have shown that testosterone therapy improves coronary blood flow (Scheuer et al., 1987). In humans, testosterone has been shown to cause a dose dependant vasodilation both in vitro and in vivo. When testosterone is instilled into the left coronary artery, vasodilation ensues and coronary flow increases (Webb et al., 1999a). More importantly, acute administration of intravenous testosterone improves exercise tolerance and reduces angina threshold in men with coronary artery disease (Rosano et al., 1999; Webb et al., 1999b). These effects were observed using supraphysiological doses, but chronic administration of low physiological replacement doses of testosterone over three months in men with chronic stable angina significantly improved exercise tolerance and angina threshold (English et al., 2000e).

As summarised in this review, several lines of evidence indicate that testosterone can exert acute vasorelaxing effects. This action of testosterone is independent of the classical androgen receptor which mediates its effects genomically, suggesting a nongenomic action. The majority of vasodilatory action of testosterone seems to be independent of the endothelium. Recent evidences suggest that the mechanism behind the vasodilatory action of testosterone is through either activation of K⁺ channels or blockade of Ca²⁺ channels in vascular muscle cells. The variance in the species and the vascular preparations used may contribute to the complex-mixture of vessel-type dependent mechanisms reported in this review.

Mechanistic studies conducted in human preparations are important to extrapolate our knowledge about vasoactive sub-stances to clinical cardiovascular situations.Therefore, to test the effects of testosterone as well as other vasoactive substances in different human arterial beds is of considerable interest (Yildiz et al., 1996a and b; Yildiz et al., 2006a and b). We have recently shown that testosterone induces vasodilation in human arteries such as IMA and RA, which are the grafts of choice for myocardial revascularization to replace diseased coronary vessels (Yildiz et al., 2005a) . We have tried testosterone on UA and observed no response. Previously, Fausett et al also reported that testosterone had no relaxant effect on human umbilical artery (1999). Obviously, additional studies in human arteries would be of interest to make clear the mechanism of vasodilatory effect of testosterone in different vascular beds.

Taken together, these findings provide evidence for the marked vasodilating effect induced by testosterone. Further studies, especially in different human vascular preparations and with cellular patch-clamp techniques, are essential to understand the exact mechanism of the effect testosterone and to fully realise the therapeutic potential of testosterone therapy in male patients with coronary artery disease.

References

Alexandersen P, Haarbo J, Byrjalsen I, Lawaetz H, Christiansen C: Natural androgens inhibit male atherosclerosis: a study in castrated, cho-lesterol-fed rabbits. Circ Res 1999; 84: 813-819 

Belfort MA, Saade GR, Suresh M, Vedernikov YP: Effects of estadiol-17 beta and progesterone on isolated human omental artery from premenopausal nonpregnant women and from normotensive and preeclamptic pregnant women. Am J Obstet Gynecol 1996; 174: 246-253 

Berridge MJ: Inositol triphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem 1987; 56: 159-193 

Cho JJ, Cadet P, Salamon E, Mantione K, Stefano GB: The nongenomic protective effects of estrogen on the male cardiovascular system: clinical and therapeutic implications in aging men) Med Sci Monit 2003 ; 9 : RA63 - 8 

Chou TM, Sudhir K, Hutchison SJ, Ko E, Amidon TM, Collins P, Chatterjee K: Testosterone induces dilation of canine coronary conductance and resistance arteries in vivo. Circulation 1996 ; 94: 2614- 2619 

Costarella CE, Stallone JN, Rutecki GW et al.: Testosterone causes direct relaxation of rat thoracic aorta. J Pharmacol Exp Ther 1996; 277: 34-39 

Crews JK, Khalil RA: Antagonistic effects of 17 beta-estradiol, proges-terone, and testosterone on Ca²⁺ entry mechanisms of coronary vaso-constriction. Arterioscler Thromb Vasc Biol 1999a; 19: 1034-1040 

Crews JK, Khalil RA: Gender-specific inhibition of Ca²⁺ entry mechanisms of arterial vasoconstriction by sex hormones. Clin Exp Pharmacol Physiol 1999b; 26: 707-715 

Darkow DJ, Lu L, White RE: Estrogen relaxation of coronary artery smooth muscle is mediated by nitric oxide and cGMP. Am J Physiol 1997; 272 (6 Pt 2):H2765-73 

Deenadayalu VP, White RE, Stallone JN, Gao X, Garcia AJ: Testosterone relaxes coronary arteries by opening the large-conductance, calcium- activated potassium channel. Am J Physiol Heart Circ Physiol 2001 ; 281: H1720-H1727 

Ding AQ Stallone JN: Testosterone-induced relaxation of rat aorta is androgen structure specific and involves K⁺ channel activation. J Appl Physiol 2001; 91: 2742-2750 

Duell PB, Bierman EL: The relationship between sex hormones and high-density lipoprotein cholesterol levels in healthy adult men. Arch Intern Med 1990; 150: 2317-2320 

English KM, Jones RD, Channer KS, Jones TH: The coronary vasodilatory effect of testosterone is mediated at the cell membrane, not by an intracellular receptor. J Endocrinol 2000c; 164: P296 

English KM, Jones RD, Jones TH, Jones TH, Morice AH, Channer KS: Gender differences in the vasomotor effects of different steroid hormones in rat pulmonary and coronary arteries. Horm Metab Res 2001; 33: 645 - 652 

English KM, Jones RD, Jones TH, Morice AH, Channer KS: Aging reduces the responsiveness of coronary arteries from male Wistar rats to the vasodilatory action of testosterone. Clin Sci 2000d; 99: 77-82 

English KM, Jones RD, Jones TH, Morice AH, Channer KS: Testosterone acts as a coronary vasodilator by a calcium antagonistic action. J Endocrinol Invest 2002 ; 25 : 455 - 458 

English KM, Mandour O, Steeds RP, Diver MJ, Jones TH, Channer KS: Men with coronary artery disease have lower levels of androgens than men with normal coronary angiograms. Eur Heart J 2000a; 21: 890-894 

English KM, Steeds R, Jones TH, Channer KS: Testosterone and coronary heart disease: is there a link? QJM 1997a; 90: 787-791 

English KM, Steeds RP, Jones TH, Diver MJ, Channer KS: Low dose transdermal testosterone therapy improves angina threshold in men with chronic stable angina. Circulation 2000b; 102: 1906-1911 

Fausett MB, Belfort MA, Nanda R: The effects of sex steroids on human umbilical artery and vein. J Soc Gynecol Investig. 1999; 6: 27-31 

Goetz CJ, Thatte HS, Prabhakar P, Cho MR, Michel T, Golan DE: Estradiol induces the calcium-dependent translocation of endothelial nitric oxide syntase. Proc Natl Acad Sci USA 1999; 96: 2788-2793 

Gul H, Yildiz O, Seyrek M, Un I: Protein Kinase C Inhibition May Con-tribute To Testosterone Induced Relaxation In Human Internal Mammary Artery. 9th Balkan Military Medical Committee, Abstract book, 2004, page:422, P-291. 

Hall J, Jones RD, Jones TH, Channer KS, Peers C: Selective inhibition of L-type Ca²⁺ channels in a7r5 cells by physiological levels of testosterone. Endocrinology. 2006; 147: 2675-2680 

Han G, Yu X, Lu L, Li S, Ma H, Zhu S, Cui X, White RE: Estrogen receptor alpha mediates acute potassium channel stimulation in human coronary artery smooth muscle cells . J Pharmacol Exp Ther 2006; 316: 1025 - 1030 

Harder DR, Belardinelli L, Sperelakis N, Rubio R, Berne RM: Differential effects of adenosine and nitroglycerin on the action potentials of large and small coronary arteries . Circ Res 1979 ; 44: 176 - 182 

HishikawaK,NakakiT,MarumoT,SuzukiH,KatoR,Saruta T: Up regulation of nitric oxide syntase by estradiol in human aortic endothelial cells. FEBS Lett 1995; 360: 291-293 

Honda H, Unemoto T, Kogo H: Different mechanisms for testosterone- induced relaxation of aorta between normotensive and spontaneously hypertensive rats. Hypertension 1999; 34: 1232-1236 

Hromadova M, Hacik T, Malatinsky E, Riecansky I: Alterations of lipid metabolism in men with hypotestosteronemia . Horm Metab Res 1991a; 23: 392-394 

Hromadova M, Hacik T, Malatinsky E, Sklovsky A, Cervenakov J, Labady F: Lipid metabolism in young males with hypotestosteronaemia and oligospermia prior to, during, and after treatment. Int Urol Nephrol 1991b; 23: 69-75 

Jones RD, English KM, Pugh PJ, Morice AH, Jones TH, Channer KS: Pul-monary vasodilatory action of testosterone: evidence of a calcium antagonistic action. J Cardiovasc Pharmacol 2002; 39: 814-823  

Jones RD, Jones TH, Channer KS: The influence of testosterone upon vascular reactivity. Eur J Endocriol 2004; 151: 29-37 

Jones RD, Pugh PJ, Hall J, Channer KS, Jones TH: Altered circulating hormone levels, endothelial function and vascular reactivity in the testicular feminised mouse. Eur J Endocrinol. 2003b; 148: 111-120 

Jones RD, Pugh PJ, Jones TH, Channer KS: Vasodilatory action of testo-sterone: a potassium-channel opening or a calcium antagonistic action? Br J Pharmacol 2003c; 138: 733-744 

Jones RD, Rowell KO, Pugh PJ, Adam IR, Vaughan R, Rocco G: Testoster-one-induced vasodilatation of isolated human mesenteric and pulmonary resistance arteries is independent of the vascular endothelium. Endocr Abstracts 2003a; 7: P66 

KhawKT, Barrett-Connor E: Blood pressure and endogenous testosterone in men: an inverse relationship. J Hypertens 1988; 6: 329-332 

Khaw KT, Barrett-Connor E: Endogenous sex hormones, high density lipoprotein cholesterol, and other lipoprotein fractions in men. Arte- rioscler Thromb 1991; 11: 489-494 

Kunz L, Ramsch R, Krieger A, Young KA, Dissen GA, Stouffer RL, Ojeda SR, Mayerhofer A: Voltage-dependent K⁺ channel acts as sex steroid sensor in endocrine cells of the human ovary. J Cell Physiol 2006 ; 167-174 

Littleton-Kearney M, Hurn PD: Testosterone as a modulator of vascular behavior. Biol Res Nurs 2004; 5: 276-285 

Messerli FH, Garavaglia GE, Schmieder RE, Sundgaard-Riise K, Nunez BD, Amodeo C: Disparate cardiovascular findings in men and women with essential hypertension. Ann Intern Med 1987; 107: 158-161 

Murphy JG, Khalil RA: Decreased [Ca(2+)](i) during inhibition of coronary smooth muscle contraction by 17beta-estradiol, progesterone, and testosterone. J Pharmacol Exp Ther 1999; 291: 44-52 

Perusquia M, Hernandez R, Morales MA, Campos MG, Villalon CM: Role of endothelium in the vasodilating effect of progestins and androgens on the rat thoracic aorta. Gen Pharmacol 1996; 27: 181-185 

Raymond V, Leung PC, Labrie F: Stimulation by prostaglandin F2 alpha of phosphatidic acid-phosphatidilinositol turnover in rat luteal cells. Biochem Biophys Res Commun 1983; 116 : 39 - 46 

Rosano GM, Leonardo F, Pagnotta P, Pelliccia F, Panina G, Cerquetani E, della Monica PL, Bonfigli B, Volpe M, Chierchia SL: Acute anti-ischemic effect of testosterone in men with coronary artery disease. Circulation 1999 ; 99 : 1666- 1670 

Sato K, Kanatsuka H, Sekiguchi N, Akai K, Wang Y, Sugimura A, Kumagai T, Komaru T, Shirato K: Effect of an ATP sensitive potassium channel opener, levcromakalim, on coronary arterial microvessels in the beating canine heart. Cardiovasc Res 1994; 28 : 1780 - 1786 

Scheuer J, Malhotra A, Schaible TF, Capasso J: Effects of gonadectomy and hormonal replacement on rat hearts. Circ Res 1987; 61: 12-19 

Scragg JL, Jones RD, Channer KS, Jones TH, Peers C: Testosterone is a potent inhibitor of L-type calcium channels . Biochem Biophys Res Comm 2004; 318 : 503 - 506 

Shimizu S, Yokoshiki H, Sperelakis N, Paul RJ: Role of voltage-dependent and Ca(2+)-activated K(+) channels on the regulation of isometric force in porcine coronary artery. J Vasc Res 2000; 37: 16-25 

Simoncini T, Genazzani AR: Non-genomic actions of sex steroid hormones. Eur J Endocrinol 2003; 148: 281-292 

Stampfer MJ, Colditz GA, Willett WC, Manson JE, Rosner B, Speizer FE, Hennekens CH: Postmenopausal estrogen thearpy and cardiovascular disease. Ten-year follow-up from the nurses . health study. N Eng L Med 1991; 325: 756-762 

Teoh H, Quan A, Leung SW, Man RY: Differantial effects of 17beta-estradiol and testosteone on the contractile responses of porcine coronary arteries. Br J Pharmacol 2000; 129: 1301-1308 

Tep-areenan P, Kendall DA, Randall MD: Mechanisms of vasorelaxation to testosterone in rat aorta. Eur J Pharmacol 2003; 465: 125-132 

Tep-areenan P, Kendall DA, Randall MD: Testosterone-induced vasore-laxation in the rat mesenteric arterial bed is mediated predominantly via potassium channels. Br J Pharmacol 2002; 135: 735-740 

Thompson J, Khalil RA: Gender differences in the regulation of vascular tone. Clin Exp Pharmacol Physiol 2003; 30 : 1 - 15 

Tosun M, Paul RJ, Rapoport RM: Intracellular Ca²⁺ elevation and con-traction due to prostaglandin F2 alpha in rat aorta. Eur J Pharmacol 1997; 340 : 203 - 208 

Valverde MA, Rojas P, Amigo J, Cosmelli D, Orio P, Bahamonde MI, Mann GE, Vergara C, Latorre R: Acute activation of Maxi-K channels (hSlo) by estradiol binding to the beta subunit . Science 1999 ; 285 : 1929 - 1931 

Webb CM, Adamson DL, de Zeigler D, Collins P: Effect of acute testosterone on myocardial ischemia in men with coronary artery disease. Am J Cardiol 1999; 83: 437-439 

Webb CM, McNeill JG, Hayward CS, de Zeigler D, Collins P: Effects of testosterone on coronary vasomotor regulation in men with coronary heart disease . Circulation 1999 ; 100 : 1690 - 1696 

White RE, Darkow DJ, Lang JL: Estrogen relaxes coronary arteries by opening BK_Ca channels through a cGMP-dependent mechanism. Circ Res 1995; 77: 936 - 942 

White RE, Han G, Maunz M, Dimitropoulou C, El-Mowafy AM, Barlow RS, Catravas JD, Snead C, Carrier GO, Zhu S, Yu X: Endothelium-independent effect of estrogen on Ca(2+)-activated K(+) channels in human coronary artery smooth muscle cells. Cardiovasc Res 2002 ; 53 : 650 - 661 

Wu FC, von Eckardstein A: Androgens and coronary artery disease. Endocr Rev 2003; 24: 183-217 

Yildiz O, Cicek S, Ay I, Demirkilic U, Tuncer M: Hypertension increases the contractions to sumatriptan in the human internal mammary artery. Ann Thorac Surg 1996 ; 62 : 1392 - 1395 

Yildiz O, Cicek S, Ay I, Tatar H, Tuncer M: 5-HT1-like receptor-mediated contraction in the human internal mammary artery. J Cardiovasc Pharmacol 1996 ; 28 : 6 - 10 

Yildiz O, Nacitarhan C, Seyrek M: Potassium channels in the vasodilating action of levosimendan on the human umbilical artery. J Soc Gynecol Investig 2006a ; 13: 312- 315 

Yildiz O, SeyrekM,GulH,UnI,YildirimV,Ozal E,Uzun M, Bolu E: Testosterone relaxes human internal mammary artery in vitro. J Car-diovasc Pharmacol 2005 ; 45 : 580 - 585 

Yildiz O, Seyrek M, Un I, Gul H, Candemir G, Yildirim V: The relationship between risk factors and testosterone-induced relaxations in human internal mammary artery. J Cardiovasc Pharmacol 2005; 45: 4-7 

Yildiz O, Seyrek M, Yildirim V, Demirkilic U, Nacitarhan C: Potassium channel-related relaxation by levosimendan in the human internal mammary artery. Ann Thorac Surg 2006b; 81: 1715-1719 

Yokoshiki H, Katsube Y, Sunagawa M, Sperelakis N: Levosimendan, a novel Ca²⁺ sensitizer, activates the glibenclamide-sensitive K⁺ channel in rat arterial myocytes. Eur J Pharmacol 1997; 333: 249-259 

Yokoshiki H, Sperelakis N: Vasodilating mechanisms of levosimendan. Cardiovasc Drugs Ther 2003; 17: 111-113 

Yue P, Chatterjee K, Beale C, Poole-Wilson PA, Collins P: Testosterone relaxes rabbit coronary arteries and aorta. Circulation 1995; 91: 1154-1160