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Endocrinology, doi:10.1210/en.2007-1288
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Endocrinology Vol. 149, No. 5 2517-2526
Copyright © 2008 by The Endocrine Society

Relaxation of Androgens on Rat Thoracic Aorta: Testosterone Concentration Dependent Agonist/Antagonist L-Type Ca2+ Channel Activity, and 5β-Dihydrotestosterone Restricted to L-Type Ca2+ Channel Blockade

Luis M. Montaño, Eduardo Calixto, Alejandra Figueroa, Edgar Flores-Soto, Verónica Carbajal and Mercedes Perusquía

Universidad Nacional Autónoma de México (M.P.), Instituto de Investigaciones Biomédicas, Departamento de Biología Celular y Fisiología, and Facultad de Medicina (L.M.M., A.F., E.F.-S.), Departamento de Farmacología, México D.F. 04510; Departamento de Hiperreactividad Bronquial (V.C.), Instituto Nacional de Enfermedades Respiratorias, México D.F. 14080; and Departamento de Neurobiología (E.C.), División de Investigación en Neurociencias, Instituto Nacional de Psiquiatria Ramón de la Fuente Muñíz, México D.F. 14370

Address all correspondence and requests for reprints to: Dr. Mercedes Perusquía, Apartado Postal 70228, México D.F. 04510. E-mail: perusqui{at}servidor.unam.mx.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgen vasorelaxing action is a subject of recent interest. We investigated the involvement of L-type voltage-operated Ca2+ channels (L-VOCCs), K+ channels, intracellular Ca2+ concentration ([Ca2+]i), and cAMP in the vasorelaxing effect of testosterone and 5β-dihydrotestosterone (5β-DHT) on rat thoracic aorta. Isolated aortic rings were used to study the vasorelaxing potency of testosterone and 5β-DHT on KCl- and noradrenaline-induced contractions. Patch-clamp was used to analyze androgen effects on Ca2+ inward and K+ outward currents. The fluorescence technique was used to evaluate [Ca2+]i in single myocytes; moreover, simultaneous measurements of [Ca2+]i and vascular contraction were evaluated. 5β-DHT was more potent than testosterone to relax KCl-induced contraction, but they were equipotent to relax noradrenaline contraction. L-type Ca2+ currents were blocked by nifedipine, both androgens, and an estrogen in a concentration-dependent manner, and the order of potency was: testosterone > nifedipine > 5β-DHT > 17β-estradiol. We observed that testosterone has different mechanism of action by the concentration range used: at nM concentrations it was a powerful L-VOCCs antagonist, whereas at µM concentrations it was observed that: 1) its Ca2+ antagonist property is reverted by increasing the L-type inward Ca2+ currents (Ca2+ agonist property); and 2) the [Ca2+]i and cAMP production was increased. The total K+ currents were unaffected by testosterone or 5β-DHT. The data show that 5β-DHT-induced vasorelaxation is due to its selective blockade on L-VOCCs (from nM to µM concentrations), but testosterone-induced vasorelaxation involves concentration-dependent additional mechanisms: acting as an L-VOCCs antagonist at low concentrations, and increasing [Ca2+]i and cAMP production at high concentrations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE PAST few years, the cardiovascular system has indeed been demonstrated to be an important target for male sex steroids (androgens). Testosterone, the principal androgen, is considered as a vasoactive steroid. Thus, in vivo and in vitro studies in animal and human vasculature have shown that testosterone causes vasorelaxation (reviewed in Refs. 1, 2, 3, 4, 5). Although isolated vessel studies commonly have reported high concentrations (10–100 µM) to detect testosterone-induced vasorelaxation, some in vivo studies have documented that testosterone also induces vasorelaxation at nM concentrations (6, 7). Controversially, some studies have also shown that testosterone may produce antivasorelaxing effects (8), particularly in long-term treatment with high doses of androgens (9, 10, 11). Therefore, the discrepancies between these studies to evoke dilatation or constriction upon vascular reactivity could be attributed to the testosterone concentrations used. However, most of the available reports have consistently shown a favorable (rather than unfavorable) effect by inducing vasorelaxation.

It is also important to highlight that the vasorelaxing effect is not privative for testosterone; actually, other androgens per se are also capable of inducing vasorelaxation, including their precursor dehydroepiandrosterone (12, 13), as well as both natural dihydro-reduced metabolites of testosterone, 5{alpha}- and 5β-dihydrotestosterone (DHT) (13, 14, 15, 16, 17). Of particular interest is that the rat thoracic aorta (5, 16) and the human umbilical artery (13) seem to be more sensitive to 5β-DHT-induced vasorelaxation than that induced by its precursors (dehydroepiandrosterone and testosterone), its 5{alpha}-reduced isomer (5{alpha}-DHT), and that produced by progestins. Likewise, 5β-DHT has also elicited greater vasorelaxation than 17β-estradiol on noradrenaline- and KCl-induced contraction in rat aorta (17). Notably, in the pulmonary vasculature, the vasorelaxing efficacy of 17β-estradiol is significantly lower than that of testosterone (18). These data raise the possibility that androgens, in particular 5β-DHT, are very effective in inducing vasorelaxation. Moreover, as a consequence of androgen-induced vasorelaxation, the ability of testosterone (19) and its 5β-reduced metabolites to induce an important vasodepressor response on rat diastolic blood pressure has been reported (20). Nevertheless, the mechanisms by which testosterone and 5β-DHT modulate the vascular tone are the subject of recent interest. At first instance, it has been admitted that androgen-induced vasorelaxation is a nongenomic mode of action (8, 16, 17, 21, 22, 23, 24) and endothelium independent (7, 14, 16, 17, 22, 24, 25, 26, 27). Thus, the available data suggest that androgens interact directly with a cellular target, resulting in a decrease of vascular tone.

The first studies with blood vessels revealed that the mechanisms involved in androgen-induced vasorelaxation may include blockade of membrane Ca2+ influx via voltage-operated calcium channels (L-VOCCs) (17, 20, 21, 25, 28) and an increased K+ channel activation (15, 23, 26). Subsequently, electrophysiological experiments in vascular cells have shown that testosterone and dehydroepiandrosterone activate calcium-sensitive potassium channels (BKCa) (14, 29). Likewise, two well-conducted studies reported that testosterone is a direct inhibitor of L-VOCCs (30) by acting as dihydropyridines to elicit direct channel blockade (31). In cardiac myocytes, testosterone, through a nongenomic effect, induces an increase of intracellular Ca2+ concentration ([Ca2+]i) via activation of a plasma membrane receptor associated with the G-protein phospholipase C/inositol 1,4,5-trisphosphate signaling pathway (32). This last evidence reveals a different action to what has been reported in vascular smooth muscle about testosterone. Admittedly, additional vasorelaxing mechanism(s) induced by testosterone must also be involved, e.g. at the level of the cGMP/protein kinase G signaling (14) and/or at the level of a receptor-activated cAMP mechanism, as suggested for progesterone-induced vasorelaxation (33).

On these bases, the present study was designed to elucidate the role of L-type Ca2+ channels, K+ channels, cytosolic Ca2+ concentration, and cAMP production in the vasorelaxing effect of testosterone and 5β-DHT, in a large range of concentrations, on the vascular reactivity of rat thoracic aorta.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Wistar rats weighing 250 g housed in our animal facility were used. The study protocol was in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health and was approved by the Institutional Animal Care and Use Committee of the Instituto Nacional de Enfermedades Respiratorias, Mexico.

Tension measurement in isolated aortic tissue
This protocol was addressed to explore the effect of androgens on KCl-induced contraction. After mild anesthesia with ether, the rats were killed by cervical dislocation, and their aortas were placed in Krebs-Henseleit solution with the following composition (mM): NaHCO3 (24.9), NaCl (119), KCl (4.74), KH2PO4 (1.18), MgSO4 (1.18), CaCl2 (2.5), and glucose (12.0); at 37 C and bubbled with 95% O2 and 5% CO2 (pH 7.4). The midthoracic region was cut into rings of approximately 1 cm in length, which were: 1) suspended horizontally, and 2) bathed in individual 10-ml tissue chambers filled with Krebs-Henseleit solution at 37 C and constantly oxygenated. Isometric tension was measured using a displacement transducer (FTO3C; Grass Instruments, Quincy, MA) and recorded on a polygraph (79; Grass Instruments). A passive resting tension of 10 mN (1 g) was maintained throughout the experiments, and the aortic rings were equilibrated for 120 min.

Because androgens induce vasorelaxation in endothelium denuded rat aorta, the aortic rings were de-endothelialized, and the absence of endothelium was pharmacologically determined when acetylcholine (20 µM) failed to induce endothelium-dependent vascular relaxation on 0.3 µM noradrenaline-induced contraction, according to the method previously described in detail (16, 17). The aortic rings were then washed with Krebs. Tissues were allowed to equilibrate for 60 min and then a contraction was evoked with high potassium solution (KCl 60 mM; Krebs solution with an equimolar substitution of 60 mM KCl and 64.7 mM NaCl). The KCl-induced contraction was repeated three times, during 30 min, before any treatment. Noncumulative concentrations (from 7.5–120 µM) of testosterone or 5β-DHT, dissolved in absolute ethanol (0.1% vol/vol), were applied during the sustained phase of tension generated by KCl. The effect of androgens at each concentration was evaluated after 10 min. Afterward, the concentration-response curves to each androgen were plotted, and the inhibitory concentration 50 (IC50; value for androgen concentration to inhibit 50% of KCl-induced contraction) was obtained. Due to the insolubility of androgens, 120 µM was the highest concentration assayed. In a separate group of experiments, aortic tissue was exposed to the equivalent ethanol solvent (0.1% vol/vol) on KCl-induced contraction. To compare the sensitivity of KCl-induced contraction to androgen-induced vasorelaxation, the same procedure was followed on the contraction induced by 0.3 µM noradrenaline.

Isolation of aortic myocytes
Aortic strips were incubated at 37 C in 5 ml Hanks’ solution containing 2 mg cysteine and 0.05 U/ml papain. After 10 min the tissue was washed with Leibovitz’s solution and then placed in a Hanks’ solution containing 1 mg/ml collagenase type I and 4 mg/ml dispase II (neutral protease) during approximately 10 min at 37 C. The tissue was gently dispersed by mechanical agitation until detached cells were observed, and enzyme activity was terminated by addition of Leibovitz’s solution, then the cells were centrifuged at 600 rpm at 20 C during 5 min. The resultant pellet was resuspended in MEM containing 10% bovine fetal serum, 2 mM L-glutamine, 10 U/ml penicillin, 10 µg/ml streptomycin, and 15 mM glucose, and plated on rounded coverslips coated with sterile rat tail collagen. Cell culture was performed at 37 C in a 5% CO2 in O2 during 24–48 h. The conventional technique for smooth muscle actin expression was used, and we obtained a high proportion of myocytes in our cultures (90% pure).

Patch-clamp studies
To investigate the mechanisms of action in the vasorelaxing effect to testosterone and 5β-DHT, we analyzed the involvement of: 1) L-type Ca2+ currents, and 2) K+ currents.

Aortic myocytes on the coverslip were submerged in a perfusion chamber (~1.5–2.0 ml/min) with the following solutions: 1) for K+ currents (mM: NaCl (130), KCl (5), CaCl2 (1.8), HEPES (10), glucose (10), MgCl2 (0.5), NaHCO3 (3), KH2PO4 (1.2), and niflumic acid (0.1; to block Cl– currents) (pH 7.4), adjusted with NaOH; and 2) for Ca2+ currents, we used Ba2+ replacing Ca2+ as the inward charge carrier (mM: NaCl (136), CsCl (6), BaCl2 (5), glucose (11), HEPES (10), and niflumic acid (0.1) (pH 7.4), adjusted with CsOH. All experiments were performed at approximately 20 C. For K+ currents, patch electrodes were filled with a solution containing (mM: potassium gluconate (140), NaCl (5), HEPES (5), EGTA (10), ATP disodium salt (5), and GTP sodium salt (0.1); pH was adjusted to 7.3 with KOH. For Ca2+ currents, patch electrodes were filled with the following solution (mM: CsCl (130), MgCl2 (5), HEPES (10), EGTA (10), ATP disodium salt (5), and GTP sodium salt (0.1) (pH 7.3), adjusted with CsOH.

The whole cell patch-clamp recordings were made using patch electrodes (2–4 M{Omega}). Voltage clamp and voltage pulse generation were regulated with an Axopatch 200A amplifier (Axon, Molecular Devices, Sunnyvale, CA). Whole cell currents were filtered at 1–5 KHz, digitized (Digidata 1200; Axon, Molecular Devices) at a simple frequency of 10 KHz, and stored on a computer for later analysis through special software (pClamp v8.0; Axon, Molecular Devices).

A series of conditioning hyperpolarizing and depolarizing pulses of potentials ranging from –70 to +50 mV were applied to the myocyte in 10-mV increments from a holding potential of –60 mV during 500 msec, 1 Hz, to observe Ca2+ currents. In the case of K+ current measuring, hyperpolarizing and depolarizing potentials ranging from –80 to +80 mV were applied in 20-mV increments from holding potential of –60 mV.

Evaluation of Ca2+ currents.
In these experiments, each cell was submitted to only one protocol of conditioning pulses. Under this condition we obtained the control group, and cells were exposed to nifedipine (0.1, 0.3, and 1 µM), testosterone (0.03, 0.1, 0.3, 1, 3.2, 10, and 32 µM), or 5β-DHT (0.1, 1, 10, and 32 µM), and as a positive control 17β-estradiol (1, 3, and 10 µM). Changes in the currents were quantified with the same protocol at maximal current peak.

Evaluation of K+ currents (IK+).
After the control protocol, 5β-DHT or testosterone (1, 10, 32, and 100 µM) was added, and changes in the currents were quantified at 400 msec. To verify that only K+ currents were recorded, some cells were incubated with 10 mM tetraethylammonium.

Ca2+ measurement in rat aorta myocytes
This technique was used to explore if testosterone or 5β-DHT induces changes in the [Ca2+]i. This methodology was also used to analyze the effect of these androgens on the L-type Ca2+ channels. Aortic myocytes were loaded with 0.5 µM fura 2 acetoxymethyl ester (fura 2) in low Ca2+ (0.1 mM) at approximately 20 C. After 1 h, cells were allowed to settle down into a heated perfusion chamber, with a glass cover in the bottom, which was mounted on an inverted microscope (Diaphot 200; Nikon, Tokyo, Japan). The cells that adhered to the glass were continuously perfused (2–2.5 ml/min) with Krebs solution at 37 C (pH 7.4).

Cells loaded with fura 2 were exposed to alternating pulses of 340 and 380-nm excitation light, and emission light was collected at 510 nm using a microphotometer (Photon Technology Intl.; PTI, Birmingham, NJ). The fluorescence acquisition rate was 0.5 sec. [Ca2+]i was calculated as previously reported (34). The Kd of fura 2 was assumed to be 386 nM (35). The mean 340/380 fluorescence ratios Rmax and Rmin were obtained as previously reported (36). Single myocytes were stimulated with 10 mM caffeine to evaluate their viability. The concentration-response curves to testosterone or 5β-DHT (10, 32, and 100 µM) were plotted. Recordings were evaluated using Felix version 1.21 PTI data acquisition and analysis software.

To corroborate the effect of testosterone and 5β-DHT on the L-type Ca2+ channels, single myocytes were stimulated with 60 mM KCl; this response was considered as a control, then the cells were perfused 15 min with normal Krebs, and immediately incubated 5 min with noncumulative concentrations of testosterone (0.032, 0.1, 1, and 10 µM) or 5β-DHT (0.1, 1, and 10 µM) and stimulated with KCl. Finally, cells were washed with Krebs, and a last KCl stimulus was done.

Simultaneous measurement of [Ca2+]i and contraction
This protocol was performed to evaluate the possible correlation between changes in the [Ca2+]i and the vasorelaxation induced by androgens. Endothelium denuded aortic rings (~0.5 cm in length) were suspended vertically in a 4-ml fluorometer cuvet of polymethacrylate, filled with Krebs solution using a special PTI adaptor, and placed inside of a PTI fluorometer. The aortic ring was fixed to the adaptor and attached to an isometric force transducer (FSG-01; Experimetria Ltd., Budapest, Hungary) connected to an analog-digital interface (PTI) via an EasyGraf recorder (model TA240; Gould Electronics, Cleveland, OH).

Preparations were equilibrated for 1 h under a resting tension of 1–1.5 g, and the tissues were stimulated with KCl during 15 min. The absence of endothelium was pharmacologically determined as mentioned previously. The tissues were then again stimulated with KCl during 30 min and washed with Krebs solution. Afterward, rings were loaded with 20 µM fura 2, 0.1% Cremophor, and 0.03% pluronic F-127 during 1 h, and subsequently stimulated with KCl during 30 min. 5β-DHT or testosterone (each at 120 µM; the highest concentration tested in isolated aortic tissue) was added during the plateau contraction. Data from fura 2 loaded tissue were obtained using a PTI fluorometer. [Ca2+]i was calculated as previously reported (36). Recordings were evaluated using Felix version 1.21.

cAMP measurement in rat aorta myocytes
On the basis that cAMP concentration is an important pathway to evoke relaxation, we decided to measure the production of this second messenger. cAMP concentration was measured through a competitive enzyme immunoassay kit (Cayman Chemical Co., Ann Arbor, MI). Aortic rings were stimulated with KCl for 30 min and then challenged with 100 µM papaverine (inhibitor of phosphodiesterase by causing elevation of cAMP levels), testosterone, or 5β-DHT (each one at 120 µM; the highest concentration tested in aortic tissue) during 30 min to record the vasorelaxing effect of the aforementioned compounds; the cAMP was then evaluated in tissues without any treatment (control group). Finally, the aortic rings were collected and stored in vials at –70 C until their study. The tissue was homogenized in 5% trichloroacetic acid in water with a homogenizer (Brinkmann Polytron Kinematica PT3100; Brinkmann Instruments, Inc., Westbury, NY) centrifuged during 10 min at 20,800 g. Trichloroacetic acid was extracted from the supernatant samples using water-saturated ether, and evaporated until dry using a centrifugal concentrator under vacuum (Speed Vac, model SC110; Savant Instruments, Holbrook, NY). Samples were reconstituted with 150 µl enzyme immunoassay buffer and read at 405 nm using a Multiskan MS photometer (Labsystems Oy, Helsinki, Finland). Acetylated cAMP concentration was expressed as pmol/mg tissue after comparing with a standard curve made with the same kit. The working range of the acetylated cAMP assay was from 0.1–10 pmol/ml.

Data analysis
To analyze Ca2+, K+ currents, and [Ca2+]i, we used the paired Student’s t test. In some experiments we used repeated measures ANOVA with the Dunnett multiple comparisons test. In isolated tissues we used the nonpaired Student’s t test. The potency of each androgen was evaluated through their IC50 values on: 1) KCl- and noradrenaline-induced contraction in blood vessels; 2) Ca2+ current; and 3) KCl-induced [Ca2+]i increment in aortic myocytes. The IC50 was calculated from every noncumulative concentration-response curve by straight-line regression. To evaluate cAMP concentrations, we used one-way ANOVA with the Dunnett multiple comparisons test. Statistical significance was set at two-tailed P < 0.05. For blood vessels, n = number of rats used in each treatment; for patch-clamp and [Ca2+]i studies, n = number of cells studies from different animals. The data are shown as the mean ± SEM.

Drugs
With the exception of nifedipine obtained from ICN Pharmaceuticals, Inc. (Costa Mesa, CA), caffeine, ouabain, H89, papaverine, mibefradil cremophor, fura 2, and the steroids were purchased from Sigma Chemical Co. (St. Louis, MO), and included: 17β-hydroxy-4-androsten-3-one (testosterone); 17β-hydroxy-5β-androstan-3-one (5β-DHT); and 1,3,5(10)-estratriene-3,17β-diol (17β-estradiol). The steroids were separately prepared as stock solution in absolute ethanol and then diluted in absolute ethanol to the concentration needed for each experiment. Each concentration was always added to the physiological medium in a final volume of 0.1% absolute ethanol (vol/vol).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Androgen effect on vasomotor activity
The data showed that in aortic rings, testosterone and 5β-DHT elicited a concentration-dependent vasorelaxing effect on KCl-induced contraction, which was immediate in onset, occurring within 1 min, and reversible after washouts (Fig. 1Go). The vasorelaxing effect of androgens at all concentrations tested was statistically different (P < 0.001) when compared with that produced by 0.1% ethanol, which relaxed no more than 1.03 ± 0.48% (n = 6). However, it is important to highlight that 5β-DHT relaxes KCl contraction more potently than testosterone; thus, the IC50 values showed that 5β-DHT (32.11 ± 0.48 µM; n = 6) was 3.15 times more potent than its precursor, testosterone (101.16 ± 7.70 µM; n = 6). Regarding the sensitivity of noradrenaline-induced contraction to androgens, the IC50 values indicated that: 1) 5β-DHT (103.49 ± 8.77 µM; n = 6) was not more potent than testosterone (84.74 ± 4.33 µM; n = 6); and 2) KCl-induced contraction was significantly (P < 0.01) more sensitive than the contraction induced by noradrenaline to 5β-DHT-induced vasorelaxation. To explore any potential interaction of testosterone and 5β-DHT on cAMP-dependent protein kinase A (PKA)-mediated phosphorylation, which is a kinase involved in the relaxation of vascular smooth muscle, an additional protocol was performed. Aortic rings were preincubated with 1 µM H89 (a nonselective PKA inhibitor) after KCl-induced contraction and 10 min before 120 µM androgen addition, resulting that their vasorelaxing response was unaffected. Furthermore, because an increased activity of the Na+/K+-ATPase pump induces hyperpolarization of the smooth muscle and, thus, promotes relaxation (37), we also analyzed any possible interaction between these androgens and the aforementioned ATPase. To this proposal an identical concentration-response curve to testosterone or 5β-DHT was obtained in the presence of 10 µM ouabain (an ATPase Na+/K+ inhibitor). Under these conditions the curves to testosterone or 5β-DHT were not significantly modified (P > 0.05).


Figure 1
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  		FIG. 1. A, Representative recordings of androgen inhibition on KCl-induced contraction; the short black line indicates the incubation time of 5β-DHT at 120 µM. Note the contraction recovery after washout (W). B, Concentration-response curves to testosterone (T) and 5β-DHT on contraction induced by KCl in aortic rings of rat. Each point represents n ≥ 6 ± SEM. *, P < 0.01.

 
Androgen effects on single aortic myocytes and aortic rings
Ca2+ currents.
In aortic myocytes, conditional pulses provoked voltage-dependent inward Ca2+ currents. The peak inward current reached maximal amplitude at 0 mV (Fig. 2Go). The calcium channel blocker nifedipine (1 µM; n = 5) almost completely abolished this Ca2+ current (89.79 ± 3.3%), corroborating that it corresponds to L-VOCCs (Figs. 2AGo and 3AGo); furthermore, some experiments revealed that the remaining Ca2+ current (10.21%) corresponds to T-type Ca2+ channel because this current was completely blocked by 10 µM mibefradil (a selective T-type Ca2+ channel antagonist). We investigated the effect of testosterone, 5β-DHT, and 17β-estradiol on L-VOCC currents. These steroids also blocked the L-type Ca2+ currents in a concentration-dependent manner (Fig. 2Go, B–D). In fact, the IC50 values showed that testosterone (IC50 = 150 nM) was three times more potent than nifedipine (IC50 = 450 nM). However, nifedipine was 4.2 times more potent than 5β-DHT (IC50 = 1.89 µM) and 8.2 times more potent than 17β-estradiol (IC50 = 3.70 µM). Thus, the rank order of potency was: testosterone > nifedipine > 5β-DHT > 17β-estradiol (Fig 3AGo). Surprisingly, testosterone from 3.1–10 µM diminished its effect as L-type Ca2+ blocker and even at 32 µM significantly increased the voltage-dependence inward Ca2+ currents (Fig. 3Go, A and B). Moreover, in the presence of nifedipine, the remaining Ca2+ current was not blocked by testosterone at 32 µM. (data not shown). Furthermore, the vehicle of nifedipine and steroids (ethanol 0.1%) was ineffective on Ca2+ currents (data not shown). In addition, these results were not different from those obtained from freshly dissociated aortic cells (data not shown).


Figure 2
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  		FIG. 2. Ca2+ currents (ICa2+) in aortic myocytes from rats. Effect of nifedipine (Nif) (A), testosterone (T) (B), 5β-DHT (C), and 17β-estradiol (17β-E2) (D) on the ICa2+ produced by a series of conditioning hyperpolarizing and depolarizing pulses of potentials ranging from –70 to +50 mV. Note that nifedipine and all the steroids used completely abolished the ICa2+. Insets correspond to example currents evoked by step depolarization from –60 to 0 mV before and during the drug applications. Each point represents the mean ± SEM of different cells. ms, msec.

 

Figure 3
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  		FIG. 3. A, Concentration-response curves to nifedipine (Nif), testosterone (T), 5β-DHT, and estradiol (17β-E2) on the Ca2+ currents (ICa2+) in isolated aortic myocytes from rats. B, Effect of testosterone, at higher concentrations, on the ICa2+ produced by a series of conditioning hyperpolarizing and depolarizing pulses of potentials ranging from –70 to +40 mV. Inset corresponds to example currents evoked by step depolarization from –60 to 0 mV before (control) and during the testosterone application. Each point represents the mean ± SEM of different cells. *, P < 0.05; **, P < 0.01.

 
K+ currents.
Aortic myocytes showed prominent noninactivating outward currents due to K+ channel activity, which increased with membrane depolarization. These currents were almost completely blocked by 10 mM tetraethylammonium, demonstrating that they corresponded to total K+ currents (IK+) (data not shown). These currents were not modified by preincubation with any concentration of testosterone or 5β-DHT (Fig. 4Go).


Figure 4
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  		FIG. 4. Effect of testosterone (T) (A) and 5β-DHT (B) on the total K+ currents (IK+) in aortic myocytes from rats. Insets correspond to typical recordings of IK+ evoked by step depolarization from –80 to 80 mV before and during the drug applications. Each point represents the mean ± SEM of different cells. ms, msec.

 
Simultaneous measurements (contraction and [Ca2+]i).
Aortic rings stimulated with 60 mM KCl produced a sustained contraction with an increment in the [Ca2+]i, indicated by the ratio of fluorescence intensity at 340 and 380 nm (Fig. 5Go). Addition of 120 µM testosterone induced a vasorelaxing response without modification of the [Ca2+]i. Interestingly, when UV light was applied, the system did not detect vasorelaxation to 120 µM 5β-DHT (data not shown). The simplest interpretation of this finding suggests that the UV light pulses could be destabilizing the 5β-DHT molecule, and, as a consequence, this blocks its vasorelaxing action.


Figure 5
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  		FIG. 5. Representative recording of simultaneous measurements of [Ca2+]i and contraction of aortic rings from rats. Testosterone (T) completely relaxed the KCl-induced contraction without modifying the [Ca2+]i.

 
To validate the aforementioned hypothesis, an additional study was performed. Therefore, the electronic structure and properties of 5β-DHT and testosterone were investigated under the UV light pulses applied, as previously described (38). The ionization potential resulted larger for 5β-DHT, the wavelength peak of UV light absorbed by testosterone was 300.5 nm, whereas 5β-DHT absorbed at 278.9 nm, and the wavelength conversion into energy resulted in 8.7 and 11.6 electro-volts for testosterone and 5β-DHT, respectively; thus, their energy values correlate as expected with the ionization potential differences (larger for 5β-DHT). On these bases, the applied UV light pulses (340/380 nm) destabilized the 5β-DHT molecule, losing its biological property to relax, whereas testosterone seems to conserve its stability under this UV light radiation, keeping its biological activity to elicit vasorelaxation. Accordingly, the vascular smooth muscle was then exposed to 440/480 UV light pulses, and 5β-DHT-induced vasorelaxation was observed.

[Ca2+]i measurements in single cells.
The resting [Ca2+]i in the aortic myocytes was 105 ± 15 nM (n = 5). Caffeine (10 mM) addition during 3 min resulted in a transient Ca2+ peak (1125 ± 275 nM). Cell stimulation with testosterone induced a small increment of [Ca2+]i, which was higher at 100 µM (Fig. 6Go). In contrast, 5β-DHT (10–100 µM; n = 5) did not induce any response in these cells (data not shown).


Figure 6
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  		FIG. 6. Effect of testosterone (T) on the [Ca2+]i in aortic myocytes from rats. A, Corresponds to example of intracellular Ca2+ increments induced by testosterone at different concentrations. All cells were stimulated with 10 mM caffeine (Caf) to evaluate its viability. B, Statistical evaluation of the testosterone effect on the [Ca2+]i. Each bar represents the mean ± SEM of n = 5 cells. *, P < 0.05 with respect to basal levels.

 
Aortic myocytes stimulated with 60 mM KCl produced a transient Ca2+ peak (110 ± 11.32 nM). This response was significantly diminished only by 0.032, 0.1, and 1 µM testosterone, and was concentration dependent. The IC50 for this effect was 141 nM. A higher concentration of testosterone (10 µM) did not modify the KCl response (Fig. 7Go). Meanwhile, 5β-DHT showed a great variability in the effect on KCl-induced [Ca2+]i increment, probably due to its inactivation by UV wavelengths as proposed previously.


Figure 7
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  		FIG. 7. Effect of testosterone on the KCl-induced [Ca2+]i increment in aortic myocytes from rats. A, Representative recordings of different concentrations of testosterone on the KCl response. B, Statistical evaluation of the testosterone effect on the KCl-induced [Ca2+]i increments. Each bar represents the mean ± SEM of n = 5 cells. *, P < 0.05; **, P < 0.01 with respect to KCl response.

 
cAMP measurement.
Figure 8Go shows that the control levels of cAMP in aortic rings was significantly increased (P < 0.05) by 100 µM papaverine (positive control), an effect clearly mimicked by 120 µM testosterone; however, 120 µM 5β-DHT did not modify the cAMP concentrations.


Figure 8
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  		FIG. 8. Effect of androgens on the cAMP concentrations in aortic smooth muscle from rats. Papaverine was used as a positive control to compare that testosterone, but not 5β-DHT, also produced an increase in the cAMP concentrations. *, P < 0.05 with respect to control group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The androgen-induced vasorelaxation has been characterized as a nongenomic (membrane) mechanism rather than the classical genomic mechanisms of steroid hormones (13, 21, 23, 24), and this process seems to be also endothelium independent (13, 14, 16, 17, 22, 24, 25, 26, 28). Testosterone and its 5β-reduced dihydro-metabolite, 5β-DHT, are vasoactive steroids that induce an acute vascular relaxation. In the present study, we found that 5β-DHT is exclusively acting as an antagonist of L-type Ca2+ channels, whereas testosterone possesses different mechanisms: 1) a dual effect on the L-type Ca2+ channels (antagonist/agonist); 2) increase in the [Ca2+]i; and 3) increment in the cAMP production. To the best of our knowledge, this is the first evidence to understand: 1) the mechanism of the acute vasorelaxing action of 5β-DHT; and 2) the controversial response of testosterone upon vascular reactivity, i.e. vasorelaxing and/or antivasorelaxing effect.

5β-DHT, at µM supraphysiological concentrations, resulted 3.15 times more potent than its precursor, testosterone, to inhibit KCl-induced contraction in isolated rat aorta. Contrastingly, in single aortic myocytes, we observed that testosterone, at concentrations less than or equal to 1 µM, was more potent than 5β-DHT to block VOCCs, and, notably, both testosterone and 5β-DHT were more potent than 17β-estradiol. Interestingly, testosterone from 3.1–10 µM diminished its potential effect as L-type Ca2+ channel blocker and even at 32 µM significantly increased the inward Ca2+ currents (Fig. 3Go, A and B), and this last effect was not through T-type Ca2+ channels because in the presence of nifedipine, the remaining T-type Ca2+ current was not affected by 32 µM testosterone. Indeed, this dual effect (antagonist/agonist) of testosterone on the activity of the L-type Ca2+ channels has not been reported before. Therefore, these findings could help us to explain why testosterone may induce antivasorelaxing (8, 9, 10, 11) or vasorelaxing (reviewed in Refs. 3 and 5) effects, which might be due to the concentration range used.

Remarkably, we have previously reported that the contractions evoked by noradrenaline or serotonin, in rat aorta (16) and human umbilical artery (13), are also sensitive to testosterone- and 5β-DHT-induced vasorelaxation. In the present study, we determined that testosterone and 5β-DHT were equipotent to relax noradrenaline-induced contraction, whereas 5β-DHT-induced relaxation on KCl-induced contraction turned out to be notably more potent than testosterone. The fact that the vasorelaxation induced by androgens on a contraction more physiologically elicited (such as by noradrenaline), reveals that their action may also involve, at least, other mechanisms, e.g. a blockade of Ca2+ influx via receptor- and store-operated Ca2+ channels, and/or the reverse mode of the Na+/Ca2+ exchanger. It is also important to highlight that 5β-DHT has also been more potent than 17β-estradiol in inhibiting contractions induced by KCl or noradrenaline in isolated rat aorta (17). Moreover, the powerful relaxing efficacy of 5β-DHT has also been confirmed on vascular tone of isolated human umbilical artery (13), as well as in nonvascular smooth muscles such as myometrium of the rat (39) and human (40), and in intestinal tissue (reviewed in Ref. 5).

The blockade of L-type Ca2+ currents by 17β-estradiol was first described in the mid-1990s (41, 42, 43), and afterwards the same phenomenon was described for progesterone (44, 45). In this regard recent studies have reported that testosterone can inhibit the activity of the native and the human recombinant L-type Ca2+ channels in the A7r5 vascular smooth muscle and HEK 293 cell lines, respectively (30), showing that the IC50 to block the L-type Ca2+ currents was 38 nM. Subsequently, the same researchers found that: 1) testosterone, at physiological concentration, as well as 5β-DHT can selectively suppress Ca2+ influx into the same vascular cell line (IC50 = 3.1 and 6.9 nM, respectively) via L-type Ca2+ channels (46); and 2) testosterone shares the same molecular requirements of the {alpha}1C subunit of L-type Ca2+ channels as dihydropyridines to elicit direct channel blockade (31). In support of this view, our data demonstrated that testosterone (≤1 µM; IC50 = 150 nM) turned out to be more efficient to block Ca2+ currents than nifedipine, a very well-known Ca2+ antagonist, and than the other two steroids, confirming that testosterone is a powerful endogenous Ca2+ channel antagonist, as recently reported. Importantly, we obtained the first direct evidence for a blockade of L-type Ca2+ currents by 5β-DHT, which elicits an acute vasorelaxing effect in isolated blood vessels. Obviously, further experiments will be required to evaluate if this androgen acts at the same site than the dihydropyridines. Our study also shows that testosterone (IC50 = 141 nM) diminishes the [Ca2+]i increment induced by KCl in rat aortic myocytes. Admittedly, our findings were observed at values outside the physiological range as compared with previous testosterone reports; however, these differences may be due to the type of cell used: primary cultured and freshly dissociated cells (present study) vs. cellular lines (30, 31, 46). In this context it has been reported that the amplitude of [Ca2+]i responses to acetylcholine and ATP in rat tracheal myocytes cultured for 10 d was dramatically increased when compared with the freshly isolated tracheal myocytes (47). Thus, it is possible that the cellular lines could be more sensitive to the blockade of VOCCs by testosterone; however, further studies are required to clarify this issue.

Our experimental data have demonstrated a different potency to decrease Ca2+ currents by testosterone and 17β-estradiol. Testosterone was more potent than 5β-DHT and 17β-estradiol (12.5 and 24.5 times, respectively) to block VOCCs in aortic myocytes. On the contrary, in aortic rings the relaxing efficacy of these androgens turned out to be different, where 5β-DHT was more potent than testosterone to inhibit KCl-induced contraction. Admittedly, this evidence generates a controversy to explain the major vasorelaxing efficacy of 5β-DHT.

Therefore, the attenuated vasorelaxing effect induced by testosterone on KCl-induced contraction, as compared with 5β-DHT, in isolated tissue might be explained by the following actions of testosterone: 1) at concentrations more than 1 µM (supraphysiological concentrations), its efficacy as antagonist on L-type Ca2+ channels diminishes, and this action is reverted by its effect as agonist up to 32 µM; and 2) induces a small increase of [Ca2+]i, which may be due to its Ca2+ agonist properties. Together, these last mechanisms represent a negative action on its vasorelaxing effect, occurring only at supraphysiological concentrations, and may explain the contradictory antivasodilatory effect. Therefore, the vasorelaxing effect of testosterone in isolated blood vessels, at pharmacological concentrations, could be explained, at least in part, by its ability to increase the cAMP production, even though the small [Ca2+]i increment, which is in line with a previous report to progesterone-induced vasorelaxation (33). Moreover, because an inhibitor of the ATPase Na+/K+ pump did not affect androgen-induced vasorelaxation, this mechanism was discarded.

Regarding the dual effect (antagonist/agonist) of testosterone on L-type Ca2+ channels, which appears to be a new pharmacological aspect of this androgen, it might explain the findings of Barbagallo et al. (48), who reported that testosterone potentiates the KCl response on the L-type Ca2+ channels in rat tail artery myocytes. Our results demonstrate that testosterone also increases [Ca2+]i in the vascular myocytes, but only at higher concentrations (up to 30 µM), whereas 5β-DHT seems to be incapable of producing this effect, even at higher concentrations (100 µM). The testosterone-mediated increment of [Ca2+]i has also been observed in cardiac myocytes, where at a very low concentration (1 nM), it induced an increase of [Ca2+]i via activation of a plasma membrane receptor associated with a G protein (32).

In addition, PKA mediates phosphorylation of phospholamban (a protein that inhibits the sarcoplasmic reticulum ATPase Ca2+ pump), which increases BKCa channel activity, and these mechanisms lead to a reduction in the [Ca2+]i to produce relaxation (49). Our data showed that H89 (a nonselective PKA inhibitor) did not modify the vasorelaxing response induced by testosterone, suggesting that its effect is a PKA independent pathway. In this context it is tempting to suggest that the K+ channels are not involved in the androgen-mediated vasorelaxation by the fact that testosterone and 5β-DHT did not modify the IK+. In support of this view, we have recently reported that BKCa, Kv, and KATP selective inhibitors are incapable of blocking androgen-induced vasorelaxation in isolated human umbilical artery (13).

Although our finding has excluded an interaction of androgens with K+ channels, several lines of experimental evidence in isolated blood vessels have suggested the involvement of these channels in testosterone-induced vasorelaxation. Thus, a selective inhibitor of KATP channels, but not other K+ channel inhibitors, reduced the relaxation induced by testosterone in the rat aorta (26) and mesenteric arterial bed (23). On the contrary, an inhibitor of Kv channels, but not other K+ channel inhibitors, may diminish testosterone-induced vasorelaxation on the rat aorta (15). Collaterally, it has also been reported that two selective inhibitors of BKCa channels attenuated the relaxation to testosterone on porcine coronary arteries (14). These discrepancies may be due to different experimental conditions, e.g. the vasoconstrictor agent used (which could generate different signaling pathways), the species differences, and/or the type of vascular bed under study. A further result from patch-clamp studies, in single porcine coronary myocytes (14), reported an increase of BKCa channel activity in treated cells (35–40 min) with 200 nM testosterone. However, this evidence should be carefully considered because when in vitro experiments are conducted using testosterone pretreatment, the rate and extent of the metabolism of this androgen should be considered. In addition, some convincing arguments against the hypothesis that testosterone-induced vasorelaxation may be through K+ channel activation have been extensively discussed in the excellent review from Jones et al. (1).

Alternatively, it is also possible that the attenuated relaxing effect of testosterone that we observe in the isolated blood vessels might result not only of different cellular sites of action but also of testosterone biotransformation into either 17β-estradiol (via the enzyme aromatase) or 5{alpha}- and 5β-DHT (via the enzymes 5{alpha}- and 5β-reductase, respectively). Indeed, testosterone can be irreversibly bioconverted into estrogens and androgens; as a consequence, an insufficient amount of testosterone interacts at the site(s) of vasorelaxant action.

In summary, the vasorelaxation induced by 5β-DHT, but not to testosterone, may be due to its selective blockade on VOCCs by acting as a pure Ca2+ antagonist from nM to µM concentrations. Contrastingly, testosterone may take different site(s) of action by the concentration range used. At nM concentrations, it is a powerful Ca2+ antagonist, but at µM concentrations, its Ca2+ antagonist property stops, and it might also play a role as a Ca2+ agonist, eliciting also increment in both [Ca2+]i and cAMP production.

Possible physiological and clinical implications
The present findings advise that the long-term treatment with pharmacological doses of testosterone, but not physiological testosterone replacement therapy, may cause adverse events on vascular tone. Notably, the beneficial importance of 5β-DHT-induced vasorelaxation, acting exclusively as a Ca2+ blocker, may be the subsequent vasodepressor response that this 5β metabolite and other 5β- reduced androgens induce in vivo (20). In support of this view, it is interesting to note that the bioconversion of testosterone into 5β-DHT is catalyzed by the enzyme 5β-reductase, and it has been reported that the activity of this enzyme is significantly lower in essential hypertensive patients compared with normotensive controls (50). In this context, we consider that 5β-reduced androgens (such as 5β-DHT) may play an important role in blood pressure regulation by inducing diminution of vascular tone. It is also tempting to suggest that the products of testosterone metabolism (estrogens and 5β-reduced androgens) might act synergistically to enhance vascular relaxation. Furthermore, other relevant characteristics should be recognized, 5β-DHT: 1) is a nonaromatizable androgen without estrogenic side effect; 2) has minimal affinity for androgen receptors and consequently is devoid of androgenic actions; and 3) acts at a nongenomic level by inducing an acute vasorelaxation. On these bases, we suggest that this 5β-dihydro-androgen might be considered as a potential component for hypertension therapy.


   Acknowledgments
 
We thank Professor Carlos Kubli-Garfias for his help with the analysis of electronic structure and properties of steroids, as well as Erika Navarrete for her assistance with organizing the digital artwork.


   Footnotes
 
This work was supported in part by a grant from Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica/Dirección General de Asuntos del Personal Académico Project No. IN221102-2.

Disclosure Statement: The authors have nothing to declare.

First Published Online February 14, 2008

Abbreviations: DHT, Dihydrotestosterone; [Ca2+]i, intracellular Ca2+ concentration; PKA, protein kinase A; L-VOCC, L-type voltage-operated Ca2+ channel.

Received September 18, 2007.

Accepted for publication February 6, 2008.


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 Introduction
 Materials and Methods
 Results
 Discussion
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