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Endocrinology, doi:10.1210/en.2005-1243
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Endocrinology Vol. 147, No. 6 2675-2680
Copyright © 2006 by The Endocrine Society

BRIEF COMMUNICATION

Selective Inhibition of L-Type Ca2+ Channels in A7r5 Cells by Physiological Levels of Testosterone

J. Hall, R. D. Jones, T. H. Jones, K. S. Channer and C. Peers

Hormone and Vascular Biology Research Group (J.H., R.D.J., T.H.J.), Academic Unit of Endocrinology, Division of Genomic Medicine, University of Sheffield, Sheffield S10 2RX, United Kingdom; Department of Cardiology (K.S.C.), Royal Hallamshire Hospital, Sheffield S10 2JF, United Kingdom; School of Medicine (C.P.), University of Leeds, Leeds LS2 9JT, United Kingdom; and Barnsley Hospital NHS Foundation Trust (T.H.J.), Barnsley S75 2EP, United Kingdom

Address all correspondence and requests for reprints to: Prof. Chris Peers, School of Medicine, University of Leeds, Leeds LS2 9JT, United Kingdom. E-mail: c.s.peers{at}leeds.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Testosterone has marked beneficial cardiovascular effects, many of which have been attributed to a vasodilatory action. However, the molecular target of testosterone underlying this effect is subject to debate. In this study, we have used microfluorimetry as a noninvasive means of examining whether testosterone could exert dilatory effects via inhibition of voltage-gated Ca2+ entry in the model vascular smooth muscle cell line, A7r5. Rises of [Ca2+]i evoked by 50 mM K+-containing solution were suppressed in a concentration-dependent manner by testosterone (IC50, 3.1 nM) and by the nonaromatizable analog, 5ß-dihydrotestosterone (IC50, 6.9 nM). The effects of testosterone were apparent in the presence of pimozide (to block T-type Ca2+ channels) but not nifedipine (to block L-type Ca2+ channels). Testosterone did not alter Ca2+ mobilization from intracellular stores by the prostaglandin analog U46619 or capacitative Ca2+ entry in cells pretreated with thapsigargin. Our results indicate that testosterone, at physiological concentrations, can selectively suppress Ca2+ entry into A7r5 cells via L-type Ca2+ channels. We suggest this effect is a likely mechanism underlying its vasodilatory actions and beneficial cardiovascular effects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ROLE OF sex hormones in cardiovascular disease has recently received growing attention. Coronary artery disease (CAD) is approximately twice as prevalent in men as it is in women (1, 2, 3) even after taking into account major risk factors, and it has been suggested that women are protected by endogenous or exogenous estrogens (4). High testosterone levels have also been regarded as a risk factor for CAD among men, although there is no published evidence to support this hypothesis apart from the finding that anabolic steroid abuse is associated with an increased incidence of myocardial infarction (5). However, studies by our group and others (6, 7) challenge this view and suggest that testosterone may be beneficial to the cardiovascular system by reducing intrinsic coronary artery tone (6), and increasing coronary blood flow (7). We have shown that men with CAD have significantly lower levels of androgens than normal controls, even after correcting for risk factors, thus implying that the low androgen status in these men may be a risk factor for the development of CAD (8).

Numerous reports describe the beneficial effects of testosterone replacement therapy in males. These include observations that chronic administration of both high-dose oral testosterone (9) or physiological transdermal testosterone (10) improves symptom scores of angina and reduces objective measures of myocardial ischemia in men with CAD. Myocardial ischemia in males with CAD has also been shown to be reduced by acute administration of iv testosterone (11, 12), whereas intracoronary infusion of physiological concentrations of testosterone is reported to increase coronary artery diameter and coronary blood flow in such individuals (7). We have also demonstrated that chronic im long-term testosterone therapy and transdermal testosterone therapy improve functional exercise capacity in men with chronic heart failure (13, 14), and acute administration of buccal testosterone therapy is associated with a significant reduction in systemic vascular resistance (15). These and other studies suggest a rapid and maintained vasodilatory effect of testosterone, which is thought to underlie the beneficial effects of testosterone in the vasculature. Other beneficial effects of testosterone on the cardiovascular system include a decrease in visceral obesity, cholesterol, insulin resistance, proinflammatory cytokines, and a prothrombotic state (16).

Testosterone-induced vasodilatation has been demonstrated in animal studies in a variety of species and vascular beds, including isolated rat mesenteric arteries (17) and rabbit coronary arteries and aorta (18), and rat pulmonary and coronary arteries (19, 20), and more recently in isolated human pulmonary and mesenteric arteries (21). Importantly, the action of testosterone is generally accepted to be independent of the classical androgen receptor, which mediates its effects genomically, suggesting a nongenomic action (18, 22, 23). This action is believed to be independent of the endothelium (18, 21, 24), of nitric oxide (18, 22), and of cyclooxygenase (22, 25). However, the mechanism behind the vasodilatory action of testosterone is still under debate and might be through either activation of K+ channels via production of cyclic guanosine monophosphate (26) or direct blockade of Ca2+ channels in vascular muscle cells; either mechanism would allow vasodilatation from reduced Ca2+ influx through voltage-gated Ca2+ channels (VGCCs; see recent review; Ref.27). Previous studies have suggested that testosterone can inhibit Ca2+ entry pathways in vascular smooth muscle, yet these reports have routinely used supraphysiological concentrations of testosterone (22, 28, 29, 30). However, our most recent study has indicated that testosterone can block both native and recombinant L-type VGCCs with IC50 values within or close to levels of circulating testosterone concentrations. These studies used whole-cell patch clamp recordings under nonphysiological conditions (31). In the present study, we use a well-characterized vascular smooth muscle cell line, A7r5, to investigate the ability of testosterone to modulate Ca2+ homeostasis under noninvasive (microfluorimetric), physiological conditions. Our results indicate that testosterone is a selective and potent inhibitor of Ca2+ entry via L-type VGCCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
A7r5 vascular smooth muscle cells, (derived from rat thoracic aorta; European Collection of Cell Cultures, Salisbury, Hants, UK) were grown in 75-cm2 medium culture flasks (Costar, Cambridge, MA) in DMEM supplemented with 10% fetal calf serum and 1% glutamax, and maintained in a humidified atmosphere at 37 C, 5% CO2 until they reached approximately 80% confluence. These monolayers were subsequently subcultured using trypsin-EDTA, and cells between passage 18 and 25 were used in all subsequent experiments. No differences were observed in cellular responsiveness between cells of different passage numbers.

Microfluorimetric measurement of [Ca2+]i
To measure cytosolic [Ca2+]i, A7r5 cells were subcultured using trypsin-EDTA, plated onto glass coverslips in 12-well plates, and grown to approximately 80% confluence. Coverslips onto which cells had grown were preincubated for 40 min at 37 C in 2 ml DMEM containing 4 µM fura 2-AM (Molecular Probes, Cambridge, Cambridgeshire, UK) in the dark, after which the DMEM containing fura 2-AM was replaced with 2 ml of control solution for 15 min to allow the fura 2-AM to de-esterify. After the incubation period, the coverslip was broken into fragments, and single fragments were transferred into an 80-µl recording chamber mounted on the stage of an inverted microscope (Nikon Diaphot 300; Nikon, Melville, NY), where cells were continuously perfused under gravity at a rate of 1–2 ml/min. Control perfusate was composed of 135 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 5 mM HEPES, 10 mM D-glucose, 2.5 mM CaCl2. (pH 7.4 with NaOH, osmolarity adjusted to 300 mosM with sucrose, 21–24 C). Ca2+-free perfusate contained 1 mM EGTA and no added CaCl2. Drugs were applied via the perfusion system, and were either dissolved in ethanol or dimethylsulfoxide. At the highest concentrations used (0.1%), neither agent altered [Ca2+]i nor altered responses to high K+-containing solutions.

Changes of [Ca2+]i were determined ratiometrically using an Improvision monochromator-based imaging system (Openlab Image Processing & Vision Co. Ltd., Coventry, UK) with alternating excitation at 340 and 380 nm (0.2 Hz) and emission at 510 nm. Regions of interest were used to restrict data collection to individual cells. All the imaging was controlled by Improvision software that included Openlab 2.2.5 (Image Processing & Vision Co. Ltd.) and operated on a Macintosh PowerPC. Changes in [Ca2+]i were calculated by determining the rise of [Ca2+]i relative to basal levels measured immediately before that particular experimental maneuver. Bar graphs indicate the peak of the calcium flux. Calibration of fluorescence ratios was performed according to Grynkiewicz et al. (32). Where relevant, results are expressed as means ± SEM, together with example traces, and statistical comparisons were made using Student’s t tests or Mann-Whitney U tests. All mean data were obtained from the number of individual cells indicated, in each case from at least three independent experiments, where experiments were replicated and multiple cells were counted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Vascular smooth muscle cells, from which A7r5 cells are derived, are known to express both L-type (Cav1.2) and T-type (Cav3.2) VGCCs (33). To investigate their potential modulation by testosterone, we examined their relative contribution to depolarization-mediated rises of [Ca2+]i by exposing cells to perfusate containing 50 mM K+ (isotonic Na+ substitution). Such a maneuver caused rapid and reversible rises of [Ca2+]i (391 ± 39 nM; e.g. Fig. 1Go, A and F), which were due entirely to Ca2+ influx, because they were almost fully abolished by removal of extracellular Ca2+ (replaced with 1 mM EGTA; e.g. Fig. 1Go, B and F).


Figure 1
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  		FIG. 1. A–E, Example recordings of [Ca2+]i from individual A7r5 cells during perfusion with 50 mM K+ solution, applied in each case for the period indicated by the solid bars. Cells were either exposed to 50 mM K+ solution alone (A), or in the absence of extracellular Ca2+ (replaced by 1 mM EGTA; B), or in the presence of nifedipine (5 µM; C) or pimozide (1 µM; D) or both drugs together (nif. + pim., E). Open bars indicate periods of exposure to each drug or solution change. Scale bars apply to all traces. F, Bar graph illustrates mean (±SEM bars) changes in [Ca2+]i (nanomolar) evoked by exposure to solution containing 50 mM K+ under the conditions illustrated in A–E. Numbers in parentheses indicate number of cells studied in each case. ***, P < 0.001 compared with control, analyzed via Student’s unpaired t test.

 
To investigate whether L-type VGCCs were being activated in the cells, a well-known blocker of L-type VGCCs, nifedipine was used. Two minutes of incubation of cells to 5 µM nifedipine in control solution and in the continuing presence of 50 mM K+ caused a significant decrease in the rise of [Ca2+]i to 188 ± 48 nM (Fig. 1Go, C and F), but, perhaps surprisingly, only 52% of the response was blocked, suggesting that T-Type VGCCs may also contribute to the response. To investigate this, cells were exposed to the T-type VGCC blocker, pimozide (34). Two minutes of exposure of cells to 1 µM pimozide before, and in the continuing presence of 50 mM K+ caused a significant suppression of the rise of [Ca2+]i to 98 nM (Fig. 1Go, D and F), as compared with responses observed in the absence of pimozide. Exposure of cells to both blockers suppressed responses to 50 mM K+ further still, compared with responses observed in the presence of nifedipine or pimozide alone (Fig. 1Go, E and F). These data suggest that both L-type and T-type Ca2+ channels contribute to the K+-evoked rises of [Ca2+]i in A7r5 cells.

Exposure of cells to testosterone (1–30 nM) for 2 min in control solution and then during exposure to 50 mM K+ solution caused a concentration-dependent decrease in the K+-evoked rise of [Ca2+]i (Fig. 2Go, A and C). The calculated IC50 of testosterone was 3.1 nM. Similar experiments conducted using the nonaromatizable form of testosterone, 5ß-dihydrotestosterone (5ß-DHT), also produced a concentration-dependent reduction in K+-evoked rises of [Ca2+]i (Fig. 2Go, B and C), indicating that testosterone was not being converted to 17ß-estradiol via aromatase to exert its inhibitory effects. The concentration-dependent inhibition of K+-evoked rises of [Ca2+]i are summarized in the bar charts of Fig. 2CGo. The calculated IC50 of DHT was 6.9 nM. It is noteworthy that increasing the testosterone concentration as high as 1 µM caused an inhibition of K+-evoked rises of [Ca2+]i of 49 ± 5% (n = 25), a degree of inhibition which was not significantly different from that evoked by 10 nM testosterone (Fig. 2CGo), suggesting that testosterone was maximally effective at 10 nM. In the presence of 5 µM nifedipine, 10 nM testosterone produced no further suppression of K+-evoked rises of [Ca2+]i (48 ± 6%, n = 34, as compared with 56 ± 7%, n = 33 (Fig. 2CGo). By contrast, testosterone (10 nM) in the presence of 1 µM pimozide reduced K+-evoked rises of [Ca2+]i significantly further (24 ± 3%, n = 31; P < 0.001) than suppression caused by pimozide alone (Fig. 2CGo). Collectively, these findings suggest that testosterone inhibits at the same site as nifedipine, i.e. the L-type Ca2+ channel, rather than T-type Ca2+ channels in these cells.


Figure 2
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  		FIG. 2. A, Example recordings of [Ca2+]i from A7r5 cells during perfusion with 50 mM K+-containing solution (for the periods indicated by the solid bars) in the additional presence of vehicle alone (0.1% ethanol) or increasing concentrations of testosterone (1–30 nM, as indicated), applied for the periods indicated by the open bars. B, As A, but cells were exposed to increasing concentrations of DHT, as indicated. Scale bars apply to traces in A and B. C, Bar graph plotting mean percent (±SEM) response evoked by 50 mM K+ solution in the presence of increasing concentrations of testosterone (open bars) or DHT (hatched bars), as indicated. Data are normalized to high K+ responses evoked in the absence of steroids. Numbers in parentheses indicate the number of cells studied in each case. **, P < 0.01; ***, P < 0.001 compared with control, analyzed via Mann-Whitney U test. Also plotted is the mean response to 50 mM K+ in the presence of both 10 nM testosterone plus 5 µM nifedipine (10 + nif; solid bar) and 10 nM testosterone plus 1 µM pimozide (10 + pim; solid bar).

 
Agonist modulation of Ca2+ signaling is important in vascular smooth muscle cell function. Endogenous ligands that activate phospholipase C can produce inositol trisphosphate to mobilize Ca2+ from intracellular stores. This, in turn, stimulates capacitative (or store depletion mediated) Ca2+ entry (CCE; Ref.35). These two means of elevating cytosolic [Ca2+] can be resolved temporally according to the protocol exemplified by the traces in Fig. 3AGo. Thus, application of the prostaglandin analog U46619, in the absence of extracellular Ca2+, caused a rise of cytosolic Ca2+ levels that was transient due to depletion of finite intracellular stores. After removal of agonist, readdition of Ca2+ to the perfusate caused a rise of [Ca2+]i due to influx via CCE. Exposure of cells to testosterone—even at the high concentration of 1 µM-–had no significant effect on these Ca2+ signals. The bar graph of Fig. 3AGo plots peak rises of [Ca2+]i caused by U46619 application in the absence and presence of extracellular Ca2+. To determine any potential effects of testosterone on CCE, an additional protocol was undertaken. Cells first were preincubated with 1 µM thapsigargin in the absence of intracellular Ca2+ to ensure complete depletion of stores, then Ca2+ was readmitted to the perfusate, resulting in marked rises of [Ca2+]i due to maximal activation of CCE (Fig. 3BGo). Testosterone again failed to modulate this CCE, even at the high concentration of 1 µM. To confirm this Ca2+ entry was via CCE, we found it to be sensitive to inhibition by the well-characterized inhibitor of CCE, SK&F96365, in a concentration-dependent manner (Fig. 3BGo).


Figure 3
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  		FIG. 3. A, left, Two example recordings of [Ca2+]i from A7r5 cells during perfusion with 1 µM U46619 in a Ca2+-free solution. U46619 was applied for the period indicated by the solid bar. For the periods indicated by the open bars, Ca2+ (2.5 mM) was readmitted to the perfusate. Experiments were conducted either in the absence (left) or presence (right) of testosterone (1 µM). Right, Bar graphs illustrate the lack of effect of testosterone on peak amplitude of transient rises of [Ca2+]i evoked by U46619 in a Ca2+-free solution (open bars) and Ca2+-containing solution (closed bars). Data are mean ± SEM, taken from the number of cells indicated in parentheses, and normalized to responses evoked by U46619 in the absence of testosterone. B, Left, CCE evoked in two example cells by reintroduction of Ca2+ to the perfusate (open bar) after 20 min of preincubation with 1 µM thapsigargin. Experiments were conducted either in the absence (left) or presence (right) of testosterone (1 µM). Right, Bar graph illustrating the lack of effect of testosterone (open bars) on peak amplitude of CCE. Data are mean percent ± SEM, taken from the number of cells indicated in parentheses, and normalized to responses evoked in the absence of testosterone. Also shown (shaded bars) are the mean inhibitory effects of SK&F96365 on CCE in these cells. ***, P < 0.001 compared with control, analyzed via Mann-Whitney U test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major finding of this study is that physiological concentrations of testosterone inhibit depolarization-mediated Ca2+ entry into the vascular smooth muscle cell line, A7r5. Thus, our IC50 value of 3.1 nM for inhibition of depolarization-mediated Ca2+ influx is within the range of estimated testosterone levels (35), and investigation of diurnal changes indicate that total testosterone ranges from approximately 15–25 nM, of which bioavailable testosterone levels range from approximately 3–5.5 nM (36). Figure 1Go provides evidence that most, if not all, of this Ca2+ influx is via L- and T-type Ca2+ channels, and two aspects of our data suggest that testosterone is selective for L-type over T-type Ca2+ channels. First, testosterone was maximally effective at 30 nM (Fig. 2Go), where it caused a degree of inhibition of Ca2+ influx remarkably similar to the effects of a supramaximal concentration of nifedipine (Fig. 1Go). Second, the observation that nifedipine (an L-type channel blocker) but not pimozide (a T-type channel blocker) blocked the effects of testosterone suggests selectivity for L-type but not T-type channel blockade in A7r5 vascular smooth muscle cells. Thus, we propose that physiological levels of testosterone (our calculated IC50 being 3.1 nM) may act by a similar mechanism to the L-type VGCC blocker nifedipine (a widely prescribed antihypertensive dihydropyridine used in the treatment of CAD and heart failure) (37) to reduce Ca2+ influx into vascular smooth muscle and, therefore, promote vasodilatation.

Therefore the Ca2+ channel blockade observed in this study could account for the vasodilatory mechanism of testosterone in the underlying smooth muscle (see introductory section).

Other studies in isolated vessel preparations have supported the idea that testosterone acts as a Ca2+ channel antagonist, by inhibiting VGCCs. It has previously been demonstrated that testosterone inhibits VGCCs in isolated rat pulmonary (22), rat coronary (30), and porcine coronary arteries (29). It has also been demonstrated that 5ß-DHT-induced vasodilatation may involve blockade of VGCCs and receptor-operated calcium channels, demonstrated in rat aorta (24, 29). However, it must be noted that all effects of testosterone or 5ß-DHT (which is nonaromatazible to estrogen) were elicited at supraphysiological concentrations. Note also that all of the aforementioned studies were carried out in isolated vessel preparations; this could account for the effects of testosterone occurring only at supraphysiological 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. Another reason for this discrepancy with previous studies in isolated vessels may be a consequence of access to smooth muscle Ca2+ channels, this is presumably 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.

We have shown recently that testosterone is a potent inhibitor of recombinant L-type calcium channels in HEK 293 cells stably transfected with the {alpha}1C subunit of the human cardiovascular L-type calcium channel (Cav1.2), using patch-clamp electrophysiological recordings. Channel blockade occurred at a concentration within the normal circulating range (31). Supraphysiological concentrations of testosterone were also shown to inhibit T-type calcium channels stably expressed in this cell line (31). We have also shown—using patch-clamp recordings—that testosterone is a blocker of native L-type calcium channels in the A7r5 cells, although at values outside the physiological range (IC50, 51 nM; Ref.31). The reasons for these discrepancies are likely to arise from differences in experimentation methodology. Patch-clamp recordings of Ca2+ currents typically rely on the use of nonphysiological recording solutions designed to prevent flow of ions through ion channels other than Ca2+ channels, and exaggerate flow of Ba2+ (rather than Ca2+) as charge carrier through VGCCs. The present study used much more physiological conditions, and used fluorometric recordings to assay Ca2+ channel activity noninvasively. Thus, the present study most likely represents an accurate picture of the potency of testosterone as a blocker of L-type Ca2+ channels.

VGCC inhibition by testosterone was rapid (within 2 min), making it unlikely to be mediated through a genomic effect. This nongenomic action of testosterone is supported by other studies, which have also shown that testosterone mediates its effects through nongenomic actions in a variety of cell types including rat osteoblasts (38), mouse lymphocytes (39), and mouse macrophages (40, 41). Furthermore, we have also shown that 5ß-DHT blocks L-type VGCCs with an IC50 of 6.1 nM, confirming that testosterone alone is blocking these channels and that it is not subsequent to its conversion to 17ß-estradiol. These findings are consistent with other reports; a recent study by Perusquia et al. (43) have shown that 3, 10, and 1000 nM 5ß-DHT significantly reduced the increase in [Ca2+]i associated with exposure to high levels of KCl in human myometrial smooth muscle cells, with 1000 nM producing the greatest effect, thus indicating that the blockade of L-type calcium channels seems to be involved in the nongenomic relaxing action of androgens (43).

In this study, we have also demonstrated that testosterone does not block inositol trisphosphate-mediated intracellular Ca2+ mobilization or CCE in the A7r5 cells. These findings are consistent with other reports in which isolated vessel preparations have been used, demonstrating that testosterone does not inhibit CCE (22). In contrast to the findings of the present study, the only other study to examine the effects of testosterone upon CCE in A7r5 cells have shown it to have an antagonistic effect upon these channels (44). In this study, 1 µM testosterone inhibited the prostaglandin F2{alpha}-mediated increase in [Ca2+]i. The reason for the contrasting results could be due to the different agonists used to activate CCE. However, in the present study we also used the well-known CCE activator, thapsigargin, which activates CCE after full depletion of intracellular Ca2+ stores via the inhibition of the calcium transporter located in the membrane of the sarcoplasmic reticulum. In the previous study by Jones et al. (44) the agonist prostaglandin F2{alpha} was used, and the precise downstream signaling mechanisms of this agonist have only been partially characterized (44).

In summary, our findings indicate that of the multiple pathways underlying Ca2+ signaling and homeostasis in vascular smooth muscle, testosterone appears specifically to target voltage-gated Ca2+ entry via L-type Ca2+ channels, whereas agonist mobilization of Ca2+ and consequent CCE remain unaffected. Importantly, testosterone was shown to exert such an effect under noninvasive, pseudophysiological conditions, and did so at physiological concentrations. These findings strongly support the concept 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 vasodilation.


   Acknowledgments
 
We are grateful for the useful comments and help of Dr. P. Aley and Dr. J. P. Boyle.


   Footnotes
 
This work was supported by the British Heart Foundation, the Sheffield Hospitals Charitable Trust, and Barnsley Hospital Endocrinology Trust Fund.

Author disclosure summary: All of the authors of this paper have nothing to declare. Thus, accordingly, J.H., R.D.J., T.H.J., K.S.C., and C.P. have nothing to declare.

First Published Online March 9, 2006

Abbreviations: CAD, Coronary artery disease; CCE, capacitative Ca2+ entry; DHT, dihydrotestosterone; VGCC, voltage-gated Ca2+ channel.

Received September 30, 2005.

Accepted for publication March 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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