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. 2003 Mar 7;138(5):733–744. doi: 10.1038/sj.bjp.0705141

The vasodilatory action of testosterone: a potassium-channel opening or a calcium antagonistic action?

Richard D Jones 1,*, Peter J Pugh 1,2, T Hugh Jones 1,3, Kevin S Channer 2
PMCID: PMC1573742  PMID: 12642373

Testosterone has classically been considered to exert a detrimental influence upon the cardiovascular system due to the high male prevalence of coronary artery disease (CAD), which even after correcting for sex-dependent differences in risk factors, remains double that in women (Wingard et al., 1983; Jousilahti et al., 1999). However, numerous cross-sectional case control studies report hypotestosteronaemia in patients with heart disease (reviewed in Alexandersen et al. (1996)) and a recent study by our group has demonstrated that men with significant CAD have markedly reduced circulating levels of testosterone (English et al., 2000c). Similarly, testosterone levels are reported to be inversely proportional to atherosclerotic risk (Khaw & Barrett-Connor, 1991; Duell & Bierman, 1990; Hromadova et al., 1991), and other risk factors for CAD including hypertension, hyperinsulinaemia, diabetes, obesity, smoking, age and an adverse thrombotic profile are all associated with hypotestosteronaemia (reviewed in English et al., 1997). Such observations contradict this traditional view and suggest that testosterone is beneficial to the cardiovascular system, at least in male individuals, and that it is a relative deficiency in circulating testosterone which is associated with coronary heart disease.

Indeed, a number of studies support this hypothesis. Research dating back to the 1940s has demonstrated a beneficial effect of testosterone therapy in male patients with angina (Hamm, 1942; Walker, 1942; Sigler & Tulgan, 1943; Lesser, 1946), although it was a further 30 years until the first placebo-controlled data was provided: Jaffe (1977) reported an improvement in myocardial ischaemia in men who had received either four or eight, weekly intramuscular testosterone injections. Subsequent studies have demonstrated that chronic administration of high dose oral testosterone (Wu & Weng, 1993), and physiological trans-dermal testosterone (English et al., 2000d), also improve symptom scores of angina and reduce objective measures of myocardial ischaemia. Furthermore, acute administration of testosterone also provides rapid improvements in myocardial ischaemia (Webb et al., 1999a; Rosano et al., 1999), prompting the suggestion that testosterone may beneficially influence coronary vascular tone. Indeed, in subsequent studies Webb et al. (1999b) demonstrated that an intra-coronary infusion of physiological concentrations of testosterone increased coronary artery diameter and coronary blood flow in male patients with CAD, consistent with a direct coronary vasodilatory action.

Numerous animal studies have also demonstrated 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 & Khalil, 1999; Crews & Khalil, 1999a; English et al., 2000b; 2002; Deenadayalu et al., 2001). The beneficial effects of testosterone upon myocardial ischaemia are attributed to this activity. 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 & Stallone, 2001) and in vessels isolated from the mesenteric (Tep-Areenan et al., 2002) and pulmonary (English et al., 2001; Jones et al., 2002a) vasculature. Furthermore, recent reports demonstrate that both acute administration of intravenous testosterone (Ong et al., 2000) and chronic administration of oral testosterone (Kang et al., 2002) enhance brachial artery responsiveness to flow and nitrate-mediated dilatation in men with CAD. Such observations demonstrate that in addition to a direct vasodilatory action, testosterone also exerts a beneficial effect upon the regulation of vascular tone controlled by the release of nitric oxide from the endothelium, and in the clinical setting in which such therapy could be employed – male patients with significant CAD.

Mechanisms of testosterone-induced vasodilatation

Involvement of the androgen receptor

Numerous studies have been conducted to elucidate the dilatory mechanism of action of testosterone. Classically testosterone is recognized to regulate cellular function via interaction with the nuclear androgen receptor (AR). Testosterone (together with the other sex hormones) is transported in the bloodstream in conjunction with a binding protein; either sex hormone binding globulin (SHBG) to which it is tightly bound, or albumin, to which testosterone has a much lower affinity. At the target cell testosterone dissociates from the binding protein, undergoes endocytosis, and in most cell types is converted to dihydrotestosterone prior to binding to the nuclear AR. The hormone-receptor complex then directly interacts with the nuclear DNA, initiating protein synthesis. This process takes hours in most instances, although some genomic effects have been identified to occur in 40 min.

The initial focus of studies investigating the vasodilatory mechanism of action of testosterone, was the degree of involvement this classical genomic signalling pathway played in the response (summarized in Table 1). The observation that testosterone-mediated vasodilatation is evident within minutes of application and is maximal by 20 min, is strongly suggestive that activation of protein synthesis is not a pre-requisite for vascular relaxation. However, more 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; Tep-Areenan et al., 2002; Jones et al., 2002a), or by the covalent linkage of testosterone analogues to albumin, thus preventing endocytosis into the smooth muscle cell (Ding & Stallone, 2001; English et al., 2000a). Furthermore, polar, non-permeable testosterone analogues have been shown to elicit greater vasodilatation than non-polar, permeable analogues (Ding & Stallone, 2001), and non-genomic testosterone analogues have also been shown to elicit greater vasodilatation than genomic-acting analogues (Yue et al., 1995; Ding & Stallone, 2001). Research from our laboratory has also demonstrated that testosterone-mediated vasodilatation is maintained in vessels isolated from testicular feminized mice, which lack a functional androgen receptor (Jones et al., 2002b).

Table 1.

Studies investigating the influence of the androgen receptor or aromatase-mediated conversion to 17β oestradiol in the vasodilatory mechanism of action of testosterone

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The observation that testosterone is able to regulate cellular activity apparently independently of the classical signalling pathway is not restricted to the vascular smooth muscle. Rapid actions of testosterone are reported in a variety of cell types (Table 2), and such observations have spurred the suggestion that testosterone may also activate alternative, non-genomic signalling pathways, and that cell surface ARs may also exist. The subject of non-genomic signal transduction is outside the scope of the present article but is extensively covered in the excellent review of Falkenstein et al. (2000). However, numerous studies utilizing a variety of diverse cell types demonstrate rapid actions of testosterone upon calcium signalling (Table 2), which clearly has implications for vascular smooth muscle.

Table 2.

Studies demonstrating rapid effects of testosterone upon calcium homeostasis in a variety of cell types, indicative of signal transduction mediated via non-genomic cell surface receptors

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Local conversion to 17β oestradiol

17β oestradiol is also recognized to elicit marked vasodilatation in a variety of vascular beds (Chester et al., 1995; Browne et al., 1999; Salom et al., 2001; English et al., 2001), and since testosterone is readily converted into 17β oestradiol via the enzyme aromatase, this presents another potential mechanism by which testosterone may induce vascular relaxation. However evidence from a number of studies (Table 1) precludes such an action: Vasodilatation to testosterone is not reduced by either aromatase inhibition (Yue et al., 1995; Tep-Areenan et al., 2002) or oestrogen receptor antagonism (Chou et al., 1996), and similar vasodilation is triggered by non-aromatizable dihydrotestosterone (Deenadayalu et al., 2001). Furthermore, in the pulmonary vasculature, the vasodilatory efficacy of 17β oestradiol is significantly lower than testosterone (English et al., 2001) and consequently aromatase-mediated conversion of testosterone into 17β oestradiol is unlikely to be involved in the response in this vascular bed.

Involvement of endogenous vasodilators

The vasodilatory action of testosterone is also reported to be independent of the release of dilator prostaglandins and of endothelial-derived dilatory agents such as endothelial derived relaxing factor (nitric oxide – NO) or endothelial derived hyperpolarising factor (summarized in Table 3).

Table 3.

Studies investigating the influence of the endothelium and dilator prostaglandins in the vasodilatory mechanism of action of testosterone

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A number of studies have investigated the involvement of dilator prostanoids in the response to testosterone by treatment with indomethacin, an inhibitor of cyclo-oxygenase, the enzyme responsible for the synthesis of the cyclic endoperoxide prostaglandin precursors from arachindonate. However, all such studies report that indomethacin has no inhibitory effect upon testosterone-mediated relaxation (Yue et al., 1995; Chou et al., 1996; Honda et al., 1999; Jones et al., 2002a). Similarly the majority of studies have demonstrated that testosterone-induced dilatation is preserved in endothelial denuded vessels (Yue et al., 1995; Honda et al., 1999; Deenadayalu et al., 2001; Perusquia et al., 1996; Perusquía & Villalón, 1999; Crews & Khalil, 1999a, b; Murphy & Khalil, 1999) or in the presence of inhibitors of nitric oxide synthase or guanylate cyclase (Yue et al., 1995; Honda et al., 1999; Deenadayalu et al., 2001; Jones et al., 2002a). Even in the few studies that do demonstrate a reduction in the efficacy of testosterone following such interventions (Chou et al., 1996; Ding & Stallone, 2001; Tep-Areenan et al., 2002), the observed attenuation is modest, with a sizeable portion of the vasodilatory response remaining. Consequently additional dilatory mechanisms must also be involved.

However, evidence does support a modulatory action for testosterone upon endothelial function. Testosterone therapy has been demonstrated to improve flow-mediated brachial artery dilatation which occurs via sheer-stress-induced release of NO (Ong et al., 2000; Kang et al., 2002). Such data indicate that whilst testosterone does not induce vasodilatation solely through direct NO release, it clearly is able to sensitize the smooth muscle to the actions of NO. Indeed, a reduced circulating testosterone profile is associated with a reduced efficacy of acetylcholine in the Tfm mouse (Jones et al., 2002b), whilst exogenous testosterone therapy is reported to improve dilatation to nitroglycerin (Kang et al., 2002). This underlying action is as yet unknown but may be at the level of the cGMP/protein kinase G axis (Deenadayalu et al., 2001).

Whilst the results of these studies demonstrate some interaction of testosterone upon endothelial cell signalling, the majority of the vasodilatory action of testosterone is effected by a direct action on the vascular smooth muscle. At present, experiments aimed at unearthing the mechanism underlying this response support two contradictory hypotheses, (i) potassium channel activation or (ii) calcium channel antagonism.

Testosterone as a potassium channel opening agent

A number of studies have provided evidence of a modulatory role for testosterone upon potassium channel function (Table 4), the first being that of Yue et al. (1995), utilizing isolated rabbit coronary arteries and thoracic aortae preconstricted with prostaglandin F2α (PGF2α). After excluding the involvement of endogenous dilatory pathways Yue et al. (1995) demonstrated that testosterone had no inhibitory effect upon the contraction induced by addition of calcium to the organ bath under depolarizing conditions, responses which were sensitive to the voltage-operated calcium channel (VOCC) blocker verapamil. Furthermore, whilst the vasodilatory action of testosterone was unaffected by incubation with the ATP-sensitive potassium channel (KATP) blocker glibenclamide, testosterone-induced vasodilatation was reduced by incubation with barium chloride (BaCl2), and in vessels pre-constricted with potassium chloride (KCl). BaCl2 acts as a non-specific inhibitor of potassium channels, and in the presence of high concentrations of extracellular potassium ions (as generated by the addition of millimolar concentrations of KCl), the residual intracellular-extracellular flux of potassium ions which occurs via membranous voltage-sensitive potassium channels (KV), calcium-sensitive potassium channels (KCa) and KATP channels is inhibited (Figure 1). Consequently under both these experimental conditions potassium channel function is compromised, in conjunction with the vasodilatory efficacy of testosterone. Yue and colleagues therefore concluded that testosterone-mediated relaxation occurred through potassium channel activation, and since the glibenclamide data precluded involvement of KATP channels, this was likely to be via activation of KCa and/or KV channels.

Table 4.

Studies investigating the influence of potassium channel modulation in the vasodilatory mechanism of action of testosterone

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Figure 1.

Figure 1

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Mechanisms of agonist-induced smooth muscle cell contraction. Smooth muscle contraction is triggered by an elevation in intracellular calcium (↑[Ca2+]i) which catalyses the interaction between the cellular actin and myosin filaments. Under resting conditions an intracellular environment with a high potassium concentration and a low calcium concentration exists with potassium ions moving along their concentration gradient to the extracellular media via calcium, voltage and ATP-sensitive potassium channels (KCa, KV and KATP) in the membrane, generating a resting membrane potential of −60 mV. (A) Addition of mM concentrations of extracellular KCl disrupts the potassium concentration gradient, preventing the intracellular to extracellular movement of potassium ions, which are instead retained intracellularly. Depolarization of the membrane potential ensues, activating voltage-operated calcium channels (VOCCs) and triggering extracellular calcium influx (Nelson & Quayle, 1995). BAY K8644 acts as a direct activator of VOCCs (Schramm et al., 1983). (B) Noradrenaline (NA) elicits smooth muscle contraction via alpha-1 adrenoceptor stimulation, which results in a G-protein coupled activation of phospholipase C (PLC) with subsequent generation of inositol triphosphate (IP3) and diacyl glycerol (DAG) from the membrane phospholipid phosphatidyl inositol biphosphate (PIP2). IP3 acts at its receptor (IP3R) located on the intracellular membrane of the sarcoplasmic and endoplasmic reticulum, triggering calcium release from these stores whereas DAG activates VOCCs through modulation of chloride channels (Criddle et al., 1996). In contrast, prostaglandin F2α (PGF2α) acts at prostanoid receptors gated directly to receptor-operated calcium channels (ROOCs) (Tosun et al., 1997). NA is also reported to activate ROCCs in smooth muscle (Tanaka et al., 2000), the proportion of the response attributed to this additional pathway varying between vascular beds. (C) Caffeine and carbachol trigger smooth muscle contraction through stimulation of calcium release from discrete intracellular stores. Carbachol activates the intracellular IP3 receptors whilst caffeine activates intracellular ryanadine receptors (RyrR) (Xu et al., 1994). (D) Thapsigargin (Thap) triggers contraction via its inhibitory action on the calcium pumps of the sarcoplasmic and endoplasmic reticulum (SERCA). Under normal conditions a cycling of calcium occurs between these intracellular stores and the cytoplasm, calcium being released from the endoplasmic reticulum and then actively pumped back into these stores via the SERCA. In the presence of thapsigargin the SERCA are irreversibly inhibited but the passive calcium release from intracellular stores is unaffected. Consequently an emptying of the intracellular calcium stores ensues, resulting in the activation of store operated calcium channels (SOCCs) and extracellular calcium entry (Treiman et al., 1998).

In contrast, activation of KATP channels is proposed to contribute to testosterone-induced dilatation in two subsequent studies. Chou et al. (1996) assessed the vasodilatory action of testosterone via the measurement of the in vivo changes in coronary blood flow (CBF) by simultaneous intravascular two-dimensional and Doppler ultrasound, and reported that the testosterone-induced increase in CBF was significantly reduced by a preceding infusion of glibenclamide (Chou et al., 1996). However the efficacy of glibenclamide was restricted to the coronary resistance vessels, which may help explain the discrepancy with the study of Yue et al. (1995) which utilized conduit coronary arteries. Honda et al. (1999) studied the vasodilatory mechanism of testosterone in thoracic aortae isolated from normal and spontaneously hypertensive rats. Again in contrast to Yue et al. (1995), vasodilatation to testosterone was significantly attenuated in the vessels harvested from normotensive animals by incubation with the KATP channel blocker glibenclamide. However in the vessels obtained from hypertensive animals, the response to testosterone was again reduced by glibenclamide, but also by the KCa channel blocker tetraethylammonium (TEA) and the KV channel blocker 4-aminopyridine (4-AP). The results of this study are interesting since they demonstrate that in the hypertensive setting testosterone influences the function of additional potassium channels, which may be a consequence of an alteration in channel expression in the disease state. However, the authors provide little explanation for the discrepancy with the study of Yue et al. (1995) in the vasodilatory mechanism of action of testosterone under normotensive conditions.

Ding & Stallone (2001) also utilized isolated rat thoracic aortae to investigate the vasodilatory mechanism of testosterone, and employed methodology very similar to Honda et al. (1999). However, Ding & Stallone (2001) demonstrated that neither incubation with glibenclamide nor the KCa channel blockers TEA and apamin had any effect on the vasodilatory response to testosterone, in vessels preconstricted with noradrenaline (NA). However, incubation with 4-AP did significantly reduce both the potency and efficacy of testosterone (as did preconstriction with KCl), and Ding & Stallone (2001) therefore proposed that testosterone acts via a KV potassium channel opening action. Whilst the study of Ding & Stallone (2001) supports the findings of Yue et al. (1995) and has provided further knowledge of the specific potassium channels modulated by testosterone, the results contradict those of Honda et al. (1999) using the same preparation, and also other work by the same investigators (Deenadayalu et al., 2001).

Taken together, these four studies provide evidence that testosterone-induced vasodilatation occurs via potassium channel opening, having a modulatory effect upon KV channels in conduit arteries and upon KATP channels in resistance vessels. However upon further scrutiny this hypothesis may not hold true.

The conclusions of Yue et al. (1995) are based on the observations that the response to testosterone is significantly reduced both by the non-specific potassium channel blocker BaCl2, and under de-polarizing conditions. However, under conditions of high extracellular potassium it is only the flow of potassium ions, rather than the channel itself, that is blocked. Since addition of testosterone to the bath has no direct effect upon the trans-membrane potassium gradient, even if it elicited a potassium channel opening action, there would still be a prohibitively large concentration and electrochemical gradient for the potassium ions to overcome, in order to reverse the membrane depolarization responsible for the vasoconstriction. Consequently one would expect the response to testosterone to be abolished under such conditions. Since the vasodilation to testosterone persists (albeit reduced by 50%) in the presence of high extracellular potassium, it is unlikely that a potassium channel opening action is solely responsible for the vasodilatation induced by testosterone in this study. Furthermore the inhibitory effect of BaCl2 is modest even at the high concentrations used.

An alternative explanation of the dilatory action of testosterone may lie with the calcium channels which are activated downstream following exposure to both KCl and PGF2α. The high extracellular potassium gradient generated by millimolar KCl triggers membrane de-polarization with subsequent activation of VOCCs (Figure 1A), whilst in contrast PGF2α is proposed to trigger extracellular calcium entry via receptor-operated calcium channels (ROCCs) (Figure 1B). The variance in the testosterone-induced dilatation following preconstriction with these agonists, could be hypothesized to be due to a differing inhibitory efficacy of testosterone upon these calcium channels. The observation that the vasodilatory action of testosterone is compromised in vessels pre-constricted with KCl, suggests that it has less of an inhibitory action upon VOCCs in this preparation. This is supported by the finding that testosterone had no effect upon verapamil-sensitive calcium contractions (Yue et al., 1995).

Similarly, in the study of Ding & Stallone (2001), despite showing a reduction in the vasodilatory efficacy of testosterone in vessels pre-constricted with KCl, significant vasodilatation still persisted. In rat thoracic aortae pre-constricted with NA, maximal dilatation to 50 μM testosterone was 69%, which was significantly reduced to 47% in vessels pre-constricted with KCl. However, the efficacy of testosterone could be considered to have three orders of activity, since significantly greater dilatation was seen in vessels preconstricted with PGF2α (89%). Consequently it is an over-simplification of this study to state that the vasodilatory action of testosterone is high when potassium channel function is maintained, and reduced when potassium channel function is compromised. The difference between the activity of testosterone in vessels preconstricted with PGF2α and NA implies a regulatory role upon calcium channel function, and also suggests a preferential antagonism of ROCCs compared to VOCCs – a proportion of the response to NA is attributed to extracellular calcium entry via VOCCs, whilst PGF2α relies solely upon ROCC activation (Figure 1B).

However, Tep-Areenan et al. (2002) have recently provided evidence of a potential potassium channel opening action for testosterone, although the complex methodology utilized in the study makes interpretation of the data somewhat difficult. Tep-Areenan et al. (2002) studied the vasodilatory action of testosterone in the isolated perfused rat mesenteric bed, at a pressure of 15 mmHg. In order to raise the pressure of the system to near to the in vivo level of 100 mmHg, the α1 adrenoceptor agonist methoxamine was added in conjunction with potassium channel modulators. Due to the varied vasoconstrictive efficacy of these agents, to ensure that all preparations were pressurized to a similar level, a wide variety of concentrations of methoxamine were employed to pre-constrict the system, sometimes in conjunction with additional agonists such as 5-hydroxy tryptamine. However the study of Tep-Areenan et al. (2002) yields some interesting findings, not in the least that under the conditions described above, testosterone produces vasodilatation within the physiological range (5–50 nM). Tep-Areenan et al. (2002) also demonstrated that the vasodilatory action of testosterone is markedly attenuated under conditions of high extracellular potassium, whilst in vessels pre-constricted with 60 mM KCl instead of methoxamine, the dilatation is abolished and a vasoconstriction is uncovered. This is indeed consistent with testosterone having a potassium channel opening action as discussed above. Subsequent experiments demonstrated that similar findings were seen in preparations exposed to the KCa channel blockers tetrabutylammonium (TBA) and charybdotoxin (ChTX), (albeit using a higher concentration of methoxamine in conjunction with 5-HT, and lower concentrations of testosterone), but not the KV channel blocker 4-AP or the KATP channel blocker glibenclamide. Tep-Areenan et al. (2002) therefore conclude that it is the calcium sensitive maxi-K channel (BKCa) which is activated upon exposure to testosterone.

Deenadayalu et al. (2001) have provided more convincing evidence of a similar action of testosterone-induced potassium channel modulation, demonstrating that the vasodilatory efficacy of testosterone is virtually abolished in isolated porcine coronary arteries preconstricted with either 80 mM KCl or the KCa channel blockers TEA or iberiotoxin (IBTX). These are almost identical findings to those of Tep-Areenan et al. (2002), but significantly, were reinforced in patch-clamping studies, utilizing individual smooth muscle cells obtained from the primary culture of these vessels. These additional electro-physiological experiments demonstrated that the primary channel regulating the electrical activity of these cells is the BKCa channel, and that testosterone acted to open this channel. In addition, the cell permeable cyclic guanosine monophosphate (cGMP) analogue, 8-bromo-cGMP, also increased BKCa channel activity, and testosterone increased the cellular production of cGMP. These findings provide convincing evidence that testosterone-induced vasodilatation occurs via the opening of BKCa channels in this preparation, and have suggested a potential effector mechanism in the production of cGMP.

Testosterone as a calcium channel antagonist

Of the studies discussed above only those of Deenadayalu et al. (2001) and Tep-Areenan et al. (2002) report the vasodilatory action of testosterone to be abolished under conditions of high extracellular potassium, consistent with the hypothesis of testosterone triggering vasodilatation via a potassium channel-opening action. The studies of Yue et al. (1995) and Ding & Stallone (2001) could be interpreted to be supportive of a calcium antagonistic action for testosterone, and numerous studies provide more direct evidence for such an action (Table 5).

Table 5.

Studies investigating the influence of calcium channel modulation in the vasodilatory mechanism of action of testosterone

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The first paper to propose a calcium antagonistic action for testosterone was that of Perusquía & Villalón (1999) who studied the vasodilatory action of 5β-dihydrotestosterone (5β-DHT) in endothelial-denuded isolated rat thoracic aortae. Perusquía & Villalón (1999) demonstrated a vasodilatory action in vessels preconstricted with KCl or NA, which was greater in KCl-preconstricted vessels.

Since 5β-DHT completely reversed the preconstriction induced by the VOCC agonist BAY K8644, Perusquía & Villalón (1999) concluded that 5β-DHT was acting to inhibit both ROCCs and VOCCs, but had a higher efficacy upon VOCCs. Consistent with this hypothesis were the subsequent observations that 5β-DHT was able to abolish calcium-induced contractions under depolarizing conditions mediated via VOCCs, and shared a similar efficacy as the VOCC blocker nifedipine in inhibiting contractions to KCl and NA (Perusquía & Villalón, 1999).

An elegant series of experiments by Raouf Khahil's group at the University of Mississippi has also provided evidence for a calcium antagonistic action of testosterone. In initial experiments Crews & Khalil (1999a) demonstrated that testosterone dilated male porcine coronary arteries preconstricted with PGF2α or KCl, but had no inhibitory action upon caffeine-induced contractions which occur via the release of calcium from ryanadine sensitive intracellular stores (Figure 1C). Testosterone also inhibited PGF2α or KCl-induced entry of 45Ca2+ into de-endothelialized porcine coronary artery strips (Crews & Khalil, 1999a). Identical findings were found in additional experiments utilizing de-endothelialized isolated rat thoracic aortic strips (Crews & Khalil, 1999b). Again testosterone dilated vessels preconstricted with NA or KCl, but had no inhibitory action upon caffeine-induced contractions (Crews & Khalil, 1999b). These studies demonstrate that the vasodilatory efficacy of testosterone is restricted to conditions where extracellular calcium entry occurs, implying blockade of ROCC and VOCCs. In subsequent work Murphy & Khalil (1999) showed a similar mechanism of action of testosterone in isolated porcine coronary artery smooth muscle cells. Testosterone pre-treatment significantly inhibited the smooth muscle cell contraction and reduced the increase in intracellular calcium [Ca2+]i associated with exposure to PGF2α or KCl but not caffeine or carbachol (an activator of IP3-mediated intracellular calcium release) (Figure 1C) (Murphy & Khalil, 1999). These studies provide convincing evidence that testosterone has little effect upon either calcium or IP3-mediated release of calcium from intracellular stores. The observation that testosterone caused marked attenuation of responses induced by KCl provides strong evidence that testosterone has an antagonistic action upon VOCCs. However, in all three papers the inhibitory efficacy and potency of testosterone was greater against responses to PGF2α or NA, which suggests that testosterone must also be efficacious in blocking ROCCs and may indeed have a higher affinity for this type of calcium channel.

Data recently published by our group are also supportive of a calcium antagonistic mechanism of action of testosterone. Utilizing isolated rat coronary arteries we have demonstrated that testosterone elicits marked vasodilatation in vessels preconstricted with PGF2α, or KCl (English et al., 2002). The vasodilatory action of testosterone was significantly attenuated in vessels pre-constricted via phorbol dibutyrate (PDBu) in calcium free saline, conditions under which only intracellular, non-calcium dependent contractile pathways are activated (English et al., 2002). Similar findings were subsequently found in isolated pulmonary arteries (Jones et al., 2002a), although as previously demonstrated, the efficacy of testosterone was significantly lower in this vascular bed (English et al., 2001). Vasodilation to testosterone was split into three orders of magnitude, dependent upon the mechanism of action of the pre-contractile agent (Jones et al., 2002a). Similar testosterone-induced dilatation occurred in vessels pre-constricted via either PGF2α, KCl or the VOCC agonist BAY K8644, with significantly lower responses being observed in vessels pre-constricted with PDBu in calcium free saline (Jones et al., 2002a). The response was abolished in vessels pre-constricted with thapsigargin which actives store-operated calcium channels (SOCCs) following depletion of the intercellular calcium stores via the inhibition of the calcium transporter located in the membrane of the sarcoplasmic reticulum (Figure 1D) (Jones et al., 2002a). Again these studies are supportive of a calcium channel antagonistic action upon VOCCs and ROCCs.

Taken together these studies demonstrate the reliance of testosterone-mediated vasodilatation upon specific mechanisms of calcium signalling. Extracellular calcium entry would appear to be a pre-requisite for a vasodilatory action since testosterone has little inhibitory activity upon contractile responses generated through the release of calcium from intracellular stores. Furthermore, the type of channel through which extracellular calcium enters the cell is also a contributory factor. Testosterone elicits marked vasodilatation is vessels pre-constricted via agonists which activate ROCCs and VOCCs, but not SOCCs.

Conclusion

Clearly the majority of studies conducted into determining the dilatory mechanism of action of testosterone can be interpreted as being supportive of a calcium antagonistic action, since the efficacy of testosterone is clearly linked to the mechanism of action of the precontractile agonist. The specific channels upon which testosterone is proposed to act and the studies which support each action are shown in Figure 2.

Figure 2.

Figure 2

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Figure summarizing the proposed sites of action of testosterone which underlie its vasodilatory activity. List of abbreviations: ↑[Ca2+]i=elevation in intracellular calcium; DAG=diacyl glycerol; IP3=inositol triphosphate; IP3R=inositol triphosphate receptor; KATP=ATP-sensitive potassium channel; KCa=calcium-sensitive potassium channel; KCl=potassium chloride; KV=voltage-sensitive potassium channel; NA=noradrenaline; PLC=phospholipase C; PGF2α=prostaglandin F2α; PIP2=phosphatidyl inositol biphosphate; ROOC=receptor-operated calcium channel; RyrR=ryanadine receptor; SOCC=store operated calcium channel; Thap=thapsigargin; VOCC=voltage-operated calcium channel.

The observation that little (Crews & Khalil, 1999a, b; Murphy & Khalil, 1999; Ding & Stallone, 2001) or no (Perusquía & Villalón, 1999; English et al., 2002; Jones et al., 2002a) reduction in the efficacy of testosterone is seen in vessels pre-constricted with KCl, demonstrate that testosterone cannot be acting to open potassium channels as has been previously suggested. Only the studies of Deenadayalu et al. (2001) and Tep-Areenan et al. (2002) report an abolition of the response to testosterone under such conditions, consistent with a potassium channel opening action. However, the findings of the report of Tep-Areenan et al. (2002) are difficult to interpret due to the inconsistent experimental conditions utilized. Therefore only the study of Deenadayalu et al. (2001) convincingly demonstrate that testosterone-mediated vasodilatation occurs via activation of potassium channels, data which are supported by the presence of electro-physiological studies and proposition of an intermediate signalling molecule.

Surprisingly few studies have utilized patch clamp methodology to examine the calcium antagonistic action of testosterone, and the absence of such studies precludes the confirmation of this mechanism of action. Only one paper, as an aside to studying the vasodilatory mechanism of action of the female sex hormone 17β oestradiol, has looked for an inhibitory action of testosterone upon calcium channel function: Nakajima et al. (1995) report that whilst 17β oestradiol attenuates calcium flux occurring via VOCCs in an embryonic rat aortic smooth muscle cell line, testosterone did not share this activity. However, until data is obtained from primary vascular smooth muscle cells, obtained from adult or at least post-natal tissue, such conclusions must be drawn with caution.

The discrepancy between the studies discussed in this review in proposing a vasodilatory mechanism for testosterone is not apparent, but the variance in the species and vascular preparations used is likely to be contributory. Taking this into consideration, the lack of mechanistic studies conducted in human preparations is likely to be significant, and clearly this is a major gap in the current literature. Such data, especially from cellular patch-clamp studies, are essential if the exact mechanism of action of testosterone is to be unearthed, and the promising results from the preliminary clinical studies of testosterone therapy in male patients with CAD are to be fully realised.

Acknowledgments

R.D. Jones is supported by the National Heart Research Fund.

Abbreviations

4-AP

4-aminopyridine

5β-DHT

5β-dihydrotestosterone

5-HT

5-hydroxytrypamine

AR

androgen receptor

BaCl2

barium chloride

BKCa

calcium sensitive maxi-potassium channel

BSA

bovine serum albumin

BAY

BAY K8644

[Ca2+]i

intracellular calcium concentration

CAD

coronary artery disease

Caff

caffeine

Carb

carbachol

CBF

coronary blood flow

cGMP

cyclic guanosine monophosphate

ChTX

charybdotoxin

DAG

diacyl glycerol

ER

oestrogen receptor

IBTX

iberiotoxin

IP3

inositol triphosphate

IP3R

inositol triphosphate receptor

KATP

ATP-sensitive potassium channel

KCa

calcium-sensitive potassium channel

KV

voltage-sensitive potassium channel

KCl

potassium chloride

Meth

methoxamine

NA

noradrenaline

NO

nitric oxide

PDBu

phorbol dibutyrate

PDBu−Ca

phorbol dibutyrate in calcium free saline

PGF2α

prostaglandin F2α

PKC

protein kinase C

PLC

phospholipase C

ROCC

receptor-operated calcium channel

RyrR

ryanadine receptor

SHBG

sex hormone binding globulin

SOCC

store-operated calcium channel

TBA

tetrabutylammonium

TEA

tetraethylammonium

Thap

thapsigargin

VOCC

voltage-operated calcium channel

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