The current study is the first to demonstrate acute in vivo vascular responses to exogenous androgen administration are dependent upon endogenous androgen status. The increased coronary conductance in the intact males compared to the castrated males was not associated with differences in androgen receptor expression. Furthermore, these experiments provide the first evidence for a physiologic effect of the androgen, epitestosterone, on the vasculature.
In the present study, nanomolar concentrations of androgen produced statistically significant increases in coronary conductance and microvessel diameter. Circulating testosterone levels in human males range from 11–44 nM [13
], though the majority of in vitro
studies have used supraphysiologic doses in the micromolar range to induce vascular dilation responses [15
]. In contrast to the majority of in vitro
data, several in vivo
studies evaluating the effects of androgen on coronary blood flow have demonstrated significant increases in flow following the infusion of physiologic concentrations of androgen [13
]. The magnitude of the androgen induced flow response, however, has varied in previous studies. Webb et al.
used 1–100 nM doses of testosterone in men with coronary artery disease to induce a 12-17% increase in coronary blood flow [13
], while Chou and colleagues demonstrated a striking 78-86% increase in coronary blood flow following the administration of 1
μM testosterone in anesthetized dogs [14
]. In the present study coronary blood flow increased ~5-8% during the testosterone infusions in the anesthetized intact males. The large variation in coronary blood flow responses in the current literature may be related to differences in the methods used to measure or calculate hemodynamic variables, effects of baseline flow on actual concentrations of androgen achieved in the coronary circulation, or perhaps species differences in response to exogenous androgen. A similar study in anesthetized pigs demonstrated increases in coronary blood flow consistent with our data following a 1
μg/L infusion of testosterone [17
]. However in contrast to the present study, prepubertal male and female pigs were used for all flow evaluations.
This study is the first to directly compare flow responses between intact and castrated males. During the in vivo
experiments in the anesthetized pigs, a main effect of castration was observed for the testosterone and 5α-dihydrotestosterone infusions. Conversely, the microvessel studies did not demonstrate statistically significant differences between the dilation responses of intact and castrated subjects to testosterone and 5α-dihydrotestosterone. The reasons for the inconsistencies between the flow and microvessel data may be related to several factors. In the in vivo
studies, androgen was infused directly into the LAD and therefore had preferential access to the endothelium. However in the microvessel studies, the androgen was administered directly into the bath and as a result contacted the smooth muscle cells of the media first. Because of the different cell types involved and possible differences in drug diffusion through the intima and media, the different experimental conditions create the potential for variable activation of dilator pathways. Another reason for the dilation differences noted in the microvessel studies may be related to the inability of an isolated vessel to mimic the complex, integrated signal conditions present in vivo
. In the microvessel studies, the effects of androgen on spontaneous tone were evaluated. Conversely, in the in vivo
studies, numerous other hormones, vasoactive and hemodynamic factors contribute to basal tone [35
], and potentially contributed to the differential response between the intact and castrated males. Furthermore, because the in vivo
studies were performed several hours prior to the microvessel studies, it is possible that androgen administration in vivo
activated genomic actions in the vasculature that persisted in the in vitro
studies. These observations indicate that in vitro
studies are useful to confirm the dilatory action of androgen on the coronary resistance vessels, however in order to determine the mechanism and clinical relevance of androgen induced dilation, in vivo
studies are critical.
In the current study the role of the androgen receptor in the mechanism of androgen induced vascular dilation was examined. Previous research has evaluated the classical androgen receptor for its potential involvement in vascular dilation. The classic androgen receptor is a nuclear receptor that, upon testosterone or dihydrotestosterone binding, initiates protein synthesis. This genomic pathway; however, typically requires several hours for completion, with some effects detectable at a minimum of 40 minutes [22
]. Because the majority of testosterone induced dilation occurs acutely, only several minutes after administration of the hormone, the predominant view is that testosterone must also be involved in a non-genomic pathway. Research has recently identified androgen receptor mediated non-genomic pathways in addition to the classic nuclear androgen receptor pathway. The non-genomic pathways induce rapid responses and involve androgen receptor interactions in the cytoplasm or at the plasma membrane of the cell, leading to the activation of various protein kinases [36
]. A recent paper by Yu and colleagues demonstrated the ability of testosterone and 5α-dihydrotestosterone to upregulate the phosphorylation of eNOS in human aortic endothelial cells through androgen receptor mediated activation of the phosphoinositide-3 kinase/Akt pathway. Specifically, the androgen receptor interacted directly with the p85α subunit of PI3-kinase. The upregulation of nitric oxide production in this study was inhibited when the androgen receptor antagonist nilutamide was administered [28
]. In the current study, a group of anesthetized, intact male pigs received in vivo
intracoronary infusions of 5α-dihydrotestosterone and epitestosterone in the presence of flutamide. The dilation induced by epitestosterone was not attenuated in the presence of flutamide; however, the response to 5α-dihydrotestosterone, an androgen with a high affinity for the androgen receptor, was diminished by flutamide. As a caveat, 10 uM flutamide has been used previously in in vitro
], but its efficacy in vivo
has not been evaluated extensively. It is also possible that androgen causes vascular dilation through multiple pathways, including the direct blockage of L-type Ca2+
channels or activation of large-conductance calcium-sensitive K+
]. As a result, only part of the dilation response may be androgen receptor mediated and susceptible to flutamide antagonism. Thus, the results from the present study indicate that the dilation response to 5α-dihydrotestosterone was largely mediated by androgen receptor activation, while the epitestosterone dilation response did not appear to involve activation of the androgen receptor.
Due to the conductance differences between the intact and castrated males in the present study and the potential involvement of the androgen receptor in these responses, androgen receptor expression was evaluated in the conductance and resistance coronary vessels. Previous work has demonstrated an up-regulation of androgen receptor mRNA and protein in vascular smooth muscle cells exposed to varying concentrations of testosterone [38
] and reduced androgen receptor staining in castrated males compared to the intact males [32
]. We therefore hypothesized that a reduction in circulating testosterone would induce a down regulation of androgen receptor expression and may be associated with the different conductance responses observed in the intact and castrated males. However in the present study androgen receptor mRNA levels and protein in the LAD and coronary microvessels were not different between the intacts and castrates. The current literature regarding post castration androgen receptor expression is indeed quite variable and tissue specific. Prostate epithelial and stromal cells of castrated male guinea pigs demonstrated a lack of androgen receptor staining four days post castration, however brain nuclei staining was unchanged following castration [39
]. In rat skeletal muscle, castration reduced androgen receptor expression in the bulbocavernosus muscle, but had no effect on the levator ani muscle [40
]; while in bovine skeletal muscle castration was associated with an increase in androgen receptor mRNA in the semitendinosus and triceps brachii but not the splenius [41
]. Interestingly, in castrated human males, androgen receptor expression was significantly increased in leukocytes compared to age matched controls [42
]. In light of these studies, androgen receptor expression in the vasculature following castration may also be location and cell type dependent. However with regard to the current study, because androgen receptor expression was quantified in both the large conduit vessels and resistance coronary microvessels, it may be concluded that differences in conductance responses between intact and castrated males during exogenous androgen administration were not due to differences in androgen receptor protein expression.
Importantly, the current study is the first to demonstrate the vascular effects of the epimer epitestosterone. Described as a weak anti-androgen, epitestosterone has been thought to be largely biologically inactive. However, coronary conductance was increased in the anesthetized, intact males during the 10 and 100 nM epitestosterone infusions, and in the microvessel experiments, vascular dilation was observed for all doses of epitestosterone. Furthermore, the magnitude of the vasomotor responses was similar to both testosterone and 5α-dihydrotestosterone. Dilation induced by epitestosterone was not attenuated by the presence of flutamide, suggesting that epitestosterone does not function through the androgen receptor to induce vascular dilation. The present study provides the first evidence of a physiologic role for epitestosterone in coronary regulation, and indicates further research is necessary to evaluate its physiological relevance and therapeutic potential.
Several limitations to the current study should be considered. The mean age of the intact males was significantly higher than the castrated males (20.83
months vs. 3.8
months). However, post hoc analysis demonstrated the in vivo
vascular responses in younger intact males (5.33
3) were not different from older intact males (27.60
5), minimizing this concern. The small group size of the conscious animal experiments and the potential effect of anesthesia on the coronary responses in the majority of the flow studies are also acknowledged. However, the conscious, chronically instrumented pigs demonstrated a similar trend for increased coronary conductance during acute androgen administration in the intact compared to the castrated males. With regard to the immunohistochemistry data, while it was demonstrated that differences did not exist in androgen receptor protein expression between the intacts and castrates, the functionality of these receptors is unknown. As a result, it is possible that intracellular signaling pathways are different between the two groups and could contribute to differences in vascular reactivity to androgen. Finally, the serum testosterone levels from all of the pigs used in the flow studies and microvessel studies were not measured, and therefore exact differences in androgen status between the two study groups are unknown. However, historical data from our lab found intact males to have testosterone levels in the range of 200
ng/dL, while castration reduces circulating testosterone by >90% [8
In conclusion, the present study is the first to examine the role of endogenous hormone status on acute vasomotor responses to physiologic concentrations of testosterone, 5α-dihydrotestosterone and epitestosterone. The data demonstrate significantly increased conductance responses in the intact males compared to the castrates, but this response was not associated with differences in androgen receptor expression as assessed by immunohistochemistry between the two groups. Additionally, the intacts demonstrated a significant dilation response to the epimer epitestosterone, which provides the first evidence for a physiologic role of this androgen on the coronary vasculature. Results from this study suggest that the efficacy of androgen therapy in hypogonadal men may be dependent on endogenous testosterone levels, and further research is necessary to evaluate the dilation pathways involved in acute vascular responses to androgen.