Abstract
Aims:
Androgen deprivation therapy is a common prostate cancer treatment which causes men to have castrate levels of testosterone. Unfortunately, most testosterone deficient patients will suffer severe erectile dysfunction (ED) and have no effective ED treatment options. Testosterone deficiency causes endothelial dysfunction and impairs penile vasodilation necessary to maintain an erection. Recent evidence demonstrates testosterone activates androgen receptors (AR) and generates nitric oxide (NO) through the Akt-endothelial NO synthase (eNOS) pathway; however, it remains unknown how castration impacts this signaling pathway.
Materials and methods:
In this study, we used a surgically castrated rat model to determine how castration impacts ex vivo internal pudendal artery (IPA) and penile relaxation through the Akt-eNOS pathway.
Key findings:
Unlike systemic vasculature, castration causes significant IPA and penis endothelial dysfunction associated with a 50% AR reduction. Though testosterone and acetylcholine (ACh) both phosphorylate Akt and eNOS, castration did not affect testosterone-mediated IPA and penile Akt or eNOS phosphorylation. Surprisingly, castration increases Ach-mediated Akt and eNOS phosphorylation but reduces the eNOS dimer to monomer ratio. Akt inhibition using 10DEBC preserves IPA eNOS dimers. Functionally, 10DEBC reverses castration induced ex vivo IPA and penile endothelial dysfunction.
Significance:
These data demonstrate how castration uncouples eNOS and provide a novel strategy for improving endothelial-dependent relaxation necessary for an erection. Further studies are needed to determine if Akt inhibition may treat or even prevent ED in testosterone deficient prostate cancer survivors.
Keywords: androgen deprivation therapy, endothelial nitric oxide synthase, internal pudendal artery, castration, Akt, erectile dysfunction
Introduction
Androgen deprivation therapy (ADT) is commonly used to eliminate gonadal testosterone production to slow prostate cancer progression and reduce tumor size [1]. Prostate cancer patients will typically receive ADT for 6–36 months [1]. A common persistent sequela of ADT is erectile dysfunction (ED), and the severity of symptoms increases with the duration of ADT. Initially, men have difficulty maintaining an erection [2]. After 9 months of ADT, only 24% of men return to sexual activity [3]. Within 2 years of initiating ADT, 86% of men report a complete loss of erections [4]. Testosterone is required for normal erectile function [5]. Autonomic neurons which innervate the penis and initiate an erection need testosterone to survive [5,6]. Vascular endothelial cells need testosterone to release nitric oxide (NO) [7]; even penile smooth muscle growth and proliferation depends on testosterone [8]. Pathological changes to penile tissue occur in the absence of testosterone and most men who undergo long term ADT do not respond to ED therapies, such as oral phosphodiesterase 5 inhibitors (PDE5i), which prevent the degradation of guanosine 3’,5’-cyclic monophosphate and improve penile vasodilation [8,9]. Unfortunately, there are no safe and effective ED treatments for testosterone deficient prostate cancer survivors [10].
ED can be attributed to endothelial dysfunction [11]. An erection is maintained by NO derived from endothelial NO synthase (eNOS) [12]. To produce NO, eNOS must maintain a homodimer conformation. Within these dimers, oxygen reduction and L-arginine oxidation produces L-citrulline and NO. Endothelial function is affected by both coupled and uncoupled eNOS. Coupled eNOS generates NO; however, superoxide is generated by uncoupled eNOS. Superoxide depletes cofactors necessary for NO production, and causes further eNOS uncoupling – thus perpetuating a pathological feedback loop [13]. Compounds such as acetylcholine (ACh) and testosterone activate eNOS via downstream phosphorylation of the serine-threonine kinase Akt which subsequently phosphorylates eNOS [14]. The Akt-eNOS signaling pathway generates NO and directly regulates erectile physiology [12].
Castration uncouples eNOS – decreasing penile NO production and causing a 4-fold increase in superoxide production [15,16]. Castrated animal penises and pre-penile vasculature, such as the internal pudendal arteries (IPA), have decreased ACh-mediated relaxation [17,18]. Castration alters the balance of coupled and uncoupled eNOS, though it remains unclear how this imbalanced ratio contributes to endothelial-dependent relaxation in the penile vasculature.
Treatments which prevent monomeric eNOS phosphorylation may reverse castration induced ED. Surprisingly, human coronary artery endothelial cells incubated in hydrogen peroxide exhibit increases in both phosphorylated Akt and eNOS [19]. Inhibiting eNOS in aortas from aged rats reduces superoxide production [20]. Senescent human umbilical vein endothelial cells (HUVEC) demonstrate higher levels of superoxide production, uncoupled eNOS, and increased phosphorylated Akt than young HUVEC cells; after treatment with the antioxidant resveratrol, superoxide production decreases, NO production increases, and phosphorylated Ser 473 Akt reduces [21]. Selective Akt inhibition may prevent superoxide generation and reverse castration induced endothelial dysfunction.
This study uses a castrated rat model to determine how castration impacts IPA and penile endothelial-dependent relaxation through the endothelial Akt-eNOS pathway. The goal is to identify Akt-eNOS dysregulated molecular mechanisms which may be feasible therapeutic targets to ameliorate or reverse low testosterone induced ED.
Methods
Castrated Rat Model
Male Sprague Dawley rats (12 weeks old, Charles River Laboratories, Wilmington, MA, USA) were housed in pairs at 22 ± 2°C on 12 hour light/dark cycles. Water and standard rodent chow were provided ad libitum. All animal procedures were conducted in accordance to guidelines set forth by the East Carolina University Brody School of Medicine Institutional Animal Care and Use Committee and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Animals were divided into control (CON: n=24) and castrated (CAST: n=24) groups; animals within the CAST group underwent bilateral surgical orchiectomies. Following surgery, CAST animals underwent a 6 week recovery period in order to mimic an extended period of androgen deprivation common among PCa patients. At 18 weeks of age and 6 weeks post-castration, all animals were anesthetized with ketamine/xylazine (75 mg/kg and 12 mg/kg respectively, intraperitoneal) and aortas, mesenteric arteries, penises and IPA were carefully excised and collected for functional and molecular experiments. Animal euthanasia was confirmed by cardiac laceration. Previously unpublished studies in our lab have confirmed that surgical castration eliminates serum testosterone (CON: 4.276±0.754, CAST: 0.144±0.02 ng/ml. n=5).
Ex Vivo Vascular Reactivity Studies
Aortas, mesenteric arteries, IPA and penises were cleaned of connective tissue and placed in ice cold Krebs solution (130mM NaCl, 4.7mM KCl, 1.18mM KH2PO4, 1.18mM MgSO4, 14.9mM NaHCO3, 5.6mM dextrose, and 1.56mM CaCl2 in distilled water). Aortas, mesenteric arteries, and IPA were cut into 2 mm rings and mounted in myographs (620M Danish Myograph Technology, Aarhus, DK) using 40 mm stainless steel wires or 200 mm pins. Each penis was segmented into individual corpus cavernosa approximately 2 mm wide by 10 mm long and mounted in muscle strip myographs (820M, Danish Myograph Technology). Myograph chambers contained aerated (95% oxygen/5% carbon dioxide) Krebs solution maintained at 37°C. A 9.8 mN resting tension was applied to cavernosal strips and aortas; mesenteric artery and IPA resting tension was determined using LabChart8 normalization software (AD Instruments, Colorado Springs, CO, USA). After a 30 minute (aorta, mesenteric artery, IPA) or 1 hour (penis) equilibration period, contractile responses to a high potassium chloride (120 mM) solution verified tissue viability.
To evaluate vascular relaxation, tissues were precontracted with phenylephrine (PE, 10−5 M: approximately 80% of maximal PE contraction) prior to concentration response curves to acetylcholine (ACh, 10−8 −10−4 M, n=8–12/group), testosterone (10−8 −10−4 M; n=8/group), dihydrotestosterone (10−8 −10−4 M, n=8/group: Cayman Chemicals, Ann Arbor, MI), or a synthetic estrogen receptor beta agonist, diarylproprionitrile (10−8 −10−4 M, n=5–8/group). In separate experiments, ACh relaxation was further assessed in the absence and presence of the Akt inhibitor 10-[4’-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride (10DEBC 10−6 M, n=6/group: Tocris, Minneapolis, MN, USA). The concentration of 10DEBC was based on previous studies [22]. Additionally, preliminary studies in IPA confirmed that this concentration did not affect the magnitude of IPA precontraction to PE. Stock solutions of testosterone, dihydrotestosterone, and diarylproprionitrile were dissolved in ethanol and concentrated one higher Log10 molar (myograph chambers contained less than 0.34% ethanol; vehicle did not impact CON or CAST vascular tone). Unless otherwise stated, all chemicals and drugs were purchased from Sigma Aldrich (St. Louis, MO, USA) and dissolved in distilled water.
Western Blot Experiments
IPA and corpus cavernosa were incubated in myograph chambers under conditions consistent with vascular reactivity studies; each chamber contained aerated (95% oxygen, 5% carbon dioxide) Krebs solution maintained at 37°C. Tissues were allowed to equilibrate for 30 minutes. Following the equilibration period, tissues were incubated in a single concentration of testosterone (10−4 M, n=8/group) or ACh (10−4 M, n=8/group) for 5 min. The contralateral IPA and corpus cavernosa of ACh incubated tissue were incubated in the presence of 10DEBC (10−6 M, n=8/group) for 30min prior to receiving ACh (10−4 M) for 5 min. Tissues were snap frozen and stored at −80°C until processed for protein extraction.
Frozen tissue was homogenized in ice cold RIPA buffer (150 mM NaCl, 1% igepal, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris at a pH of 7.35) containing a protease and phosphatase inhibitor cocktail (Halt: Thermo Fisher). Homogenates were then centrifuged at 16,000 x g for 15 minutes. Protein concentration was determined using bicinchoninic acid (BCA) protein assay. Unless otherwise indicated, proteins (20 ug) were separated under reduced conditions on 4–12% Bis-Tris SDS polyacrylamide gels (Thermo Fisher, Waltham, MA, USA). Electrophoresis was conducted at a constant 80V for 2 hours and gels were transferred onto nitrocellulose membranes. For eNOS dimer assessment, proteins were separated under non-reducing conditions. Membranes were blocked at room temperature for 1 hour in Odyssey blocking buffer formulated in tris buffered saline (TBS) (#927–50000, LI-COR, Lincoln, Nebraska, USA).
Membranes were probed overnight at 4°C for primary antibodies against: androgen receptor (AR) (1:500, Abcam #133273), pS1177-eNOS (1:250, BD Biosciences #612393), pS473-Akt (1:500, Cell Signaling Technology #4060S), eNOS (1:500, Abcam #199956), Akt (1:500, Cell Signaling Technology #4691S), and actin (1:1,000, Sigma #A5060). Membranes probed for pS1177-eNOS and pS473-Akt were stripped and imaged to confirm stripping buffer effectiveness (images not shown; NewBlot Nitro, LI-COR). Following stripping, the same membranes were reprobed with total eNOS and total Akt, respectively. Rat testes homogenate was used as a positive loading control to validate the androgen receptor antibody, and both HUVEC cells and lysate from rat aortas incubated in ACh (10−4M) were used to validate eNOS and Akt antibodies. All antibodies were dissolved in TBS containing 0.1% Tween and 5% bovine serum albumin. Secondary antibodies (donkey anti-rabbit 800cw (1:5,000, LI-COR #926–32213) or donkey anti-mouse 800cw (1:5,000, LI-COR #926–32213) were incubated for 1 hour at room temperature. Proteins were detected using LI-COR Odyssey CLx imaging system.
Statistical Analysis
Myograph data analysis was conducted using LabChart8 software, and all data are represented as mean ± standard error of the mean (SEM). Vascular relaxation is reported as a percent decrease from the PE precontraction. Statistical significance was determined by two-way ANOVAs with Bonferroni post hoc t-tests and noted for all p-values ≤0.05 (Prism 5, GraphPad, San Diego, CA, USA).
Western blot data was analyzed using ImageJ (National Institute of Health). For membranes containing two groups, protein expression statistical significance was determined by unpaired t-tests (Prism 5). One-way ANOVAs with Tukey post hoc t-tests were used to determine statistical significance of membranes containing four groups and paired t-tests determined statistical significance between tissues incubated in ACh ± 10DEBC. Data are represented as mean ± SEM and statistical significance is noted for all p-values ≤0.05.
Results
Castration causes IPA and penile endothelial dysfunction without impacting the systemic vasculature
In our animal model, castration does not impact systemic endothelial-dependent relaxation. Aortas and mesenteric arties from CON and CAST animals demonstrate comparable ACh-mediated relaxation (Figure 1A,B). Though castration does not impact systemic endothelial function, endothelial-dependent relaxation is significantly reduced in CAST IPA and penis (Figure 1C,D).
Figure 1: Castration impairs IPA and penis endothelial relaxation without impacting systemic vasculature.
ACh-mediated relaxation does not change in CAST aortas (A) or mesenteric arteries (B). Castration reduces ACh-mediated IPA relaxation and penile relaxation (C). Data presented as mean ± SEM. Aorta and mesenteric artery n=8/group. IPA and penis n=12/group. *p≤0.05 vs CON using two-way ANOVA with Bonferroni post hoc test.
IPA and penile AR protein expression decreases following castration
Testosterone-mediated eNOS phosphorylation is AR-dependent [14]. AR populations may help determine why IPA and penis are more susceptible to castration induced endothelial dysfunction than the systemic vasculature. In the current study, AR protein expression is undetectable in aortic and mesenteric vasculature (Figure 2A). AR are moderately expressed in IPA; however, penile AR protein expression is much higher and comparable to testicular AR protein expression (Figure 2A). Castration significantly reduces both IPA (Figure 2B,D) and penile (Figure 2C,E) AR expression by approximately 50%.
Figure 2: Castration reduces IPA and penis AR.
AR protein expression was undetectable in systemic vasculature, yet IPA and penile AR are highly expressed (A). Castration reduces AR expression in IPA (B,D) and penis (C,E). Data presented as mean ± SEM. For all groups, n=8. *p≤0.05 vs CON using unpaired t-test.
Castration decreases penile relaxation to testosterone and dihydrotestosterone
Despite an approximate 50% AR reduction, testosterone-mediated IPA relaxation does not change following castration (Figure 3A). In contrast, CAST penile relaxation to testosterone is significantly lower than CON (Figure 3B). Castration has similar effects on IPA and penile dihydrotestosterone-mediated relaxation. IPA dihydrotestosterone relaxation is not changed with castration (Figure 3C), yet penile relaxation to dihydrotestosterone is impaired following castration (Figure 3D). Dihydrotestosterone can be further metabolized into estrogens with an affinity for the estrogen receptor beta. To investigate possible estrogen receptor contributions, relaxation to a synthetic estrogen receptor beta agonist diarylproprionitrile was assessed. CON and CAST IPA both relax nearly %80 to diarylproprionitrile (Figure 3E) yet diarylproprionitrile does not cause penile relaxation in either group (Figure 3F).
Figure 3: Only penile AR-dependent relaxation decreases following castration.
Castration does not impact testosterone-mediated IPA relaxation (A); however, castration significantly reduces penile relaxation to testosterone (B). Dihydrotestosterone-mediated IPA relaxation is also unaffected (C). Castration reduces penile dihydrotestosterone-mediated relaxation (D). Diarylproprionitrile-mediated IPA relaxation is not impacted (E). Diarylproprionitrile does not induce penile relaxation (F). Data presented as mean ± SEM. Diarylproprionitrile n=5/group. For all other drugs, n=8/group. *p≤0.05 vs CON using two-way ANOVA and Bonferroni post hoc test.
Increased Akt and eNOS phosphorylation in castrated tissue incubated with ACh, but not testosterone
Since both testosterone and ACh phosphorylate both Akt and eNOS to generate NO, the IPA and penises were incubated in either drug before performing Western blots to assess protein phosphorylation. Testosterone incubated IPA display no changes in either eNOS (Figure 4A,B) or Akt phosphorylation following castration (Figure 4A,C). Even though CAST penises show decreased relaxation to testosterone, both eNOS (Figure 5A,B) and Akt phosphorylation (Figure 5A,C) remain unchanged in CAST penises. In ACh incubated IPA, castration increases both eNOS (Figure 6A,B) and Akt phosphorylation (Figure 6A,C). Castration also increases Ach-mediated penile eNOS (Figure 7A,B) and Akt phosphorylation (Figure 7A,C).
Figure 4: Castrated IPA exhibit no changes in testosterone-mediated eNOS and Akt phosphorylation.
In the IPA, testosterone (10−4 M) mediated eNOS phosphorylation is not impacted by castration (A,B). Likewise, Akt phosphorylation is not different (A,C). Data presented as mean ± SEM. n=8 per group.
Figure 5: Castration does not alter penile testosterone-dependent eNOS and Akt phosphorylation.
Penile testosterone (10−4 M) mediated phosphorylation of eNOS (A) and Akt (B) is not different following castration. Data presented as mean ± SEM. n=8 per group.
Figure 6: Castrated IPA demonstrate higher ACh-mediated eNOS and Akt phosphorylation than controls.
CAST IPA incubated in ACh (10−4 M) exhibit significantly higher eNOS phosphorylation than CON (A,B). Parallel increases in Akt phosphorylation in CAST IPA are also evident (A,C). Data presented as mean ± SEM. n=8 per group. *p≤0.05 vs CON using unpaired t-test.
Figure 7: Penis exhibits higher ACh-mediated eNOS and Akt phosphorylation after castration.
Penile eNOS phosphorylation is upregulated in CAST ACh (10−4 M) incubated penises (A,B). CAST ACh incubated penises demonstrate higher Akt phosphorylation than CON (A,C). Data presented as mean ± SEM. n=8 per group. *p≤0.05 vs CON using unpaired t-test
Castration induces IPA eNOS uncoupling which is reversed with Akt inhibition
The coupled or dimeric form of eNOS is required for NO production while the monomeric or uncoupled form of eNOS generates superoxide. Castration reduces the eNOS dimer:monomer ratio approximately 65% in the IPA (Figure 8). Incubating the IPA with the Akt inhibitor, 10DEBC, prior to activation with ACh and restores the CAST dimer:monomer ratios back to CON values (Figure 8). Similarly, the penile eNOS dimer:monomer is also reduced approximately 70% by castration (Figure 9). However, incubation with 10DEBC does not redimerize CAST penile eNOS which remains 50% lower compared to CON (Figure 9).
Figure 8: Akt inhibition preserves IPA eNOS dimers during ACh activation.
In ACh (10−4 M) incubated IPA, castration reduces the eNOS dimer:monomer ratio. Akt inhibition with 10DEBC (10−6 M) preserves the IPA dimer:monomer ratio. Data presented as mean ± SEM. n=8 per group. *p≤0.05 between selected groups using one-way ANOVAS with Tukey post hoc t-tests and paired t-tests for tissues incubated in ACh ± 10DEBC.
Figure 9: Castration lowers penile eNOS dimer:monomer ratios after ACh activation.
Castration reduces the eNOS dimer:monomer in ACh (10−4 M) incubated penises. 10DEBC (10−6 M) has no effect on eNOS dimerization. Data presented as mean ± SEM. n=8 per group. *p≤0.05 between selected groups using one-way ANOVAS with Tukey post hoc t-tests and paired t-tests for tissues incubated in ACh ± 10DEBC.
Akt inhibition enhances ACh relaxation in castrated IPA and penises
CAST animals exhibit decreased IPA and penile ACh-mediated relaxation. Tissue bath experiments were performed to assess ACh relaxation in the same tissues after incubation with the Akt inhibitor, 10DEBC. Akt inhibition significantly increases IPA CAST relaxation to CON values; interestingly, both CON and CAST relaxation significantly increases (Figure 10A). In the penis, Akt inhibition increases CAST endothelial-dependent relaxation, but has no effect on CON relaxation (Figure 10B).
Figure 10: Akt inhibition reverses castration induced IPA and penis endothelial dysfunction.
Castration reduces ACh-mediated endothelial-dependent relaxation is reduced in CAST IPA (A) and CAST penis (B). 10DEBC (10−6 M) incubation increases ACh relaxation in the IPA (A). Similarly, 10DEBC incubation improves CAST penile ACh relaxation (B). Data presented as mean ± SEM. n=6/group. *p≤0.05 vs CON. #p≤0.05 vs CAST using two-way ANOVA with Bonferroni post hoc test.
Discussion
Our animal model of castration demonstrates deleterious effects to both IPA and penile vasorelaxation contributing to ED. Testosterone regulates endothelial NO production through AR activation [14]. Interestingly, testosterone also regulates multiple vasodilatory signaling pathways besides endothelial NO production. The direct effects of testosterone and multiple testosterone metabolites provide further insight into how castration dysregulates IPA and penile vasorelaxation. Our goal was to understand if these observations are related to endothelial NO production, specifically through the Akt-eNOS pathway. These findings provide new insight into how castration causes pathological IPA and penis changes in testosterone-dependent signaling pathways.
Castration is known to increase the risk of cardiovascular disease [23]. Exact mechanisms regulating this pathology are unclear and a source of much debate. Systemic endothelial function is regulated by testosterone as well as estrogens [24]. Like testosterone, estrogens are capable of phosphorylating eNOS through Akt-dependent mechanisms [25]. Interestingly, castrated rabbit aortas have significantly less estrogen receptors, but AR populations are not affected [26]. This suggests estrogens may have a disproportionately larger influence over systemic endothelial function than testosterone. Aortic endothelium from castrated rats is coarse and contains numerous leukocyte adhesions [27]. In the current study, AR expression in systemic vasculature was too low for detection. Still, endothelial dysfunction was not evident within systemic vasculature following castration. This is not a surprising observation given the comparative lack of AR in systemic vasculature. Without any compounding effects of systemic endothelial dysfunction, IPA and penile endothelial dysfunction are likely a direct result of castration. The high IPA and penis AR protein concentrations suggest testosterone may be a key regulator of endothelial function.
The pro-erectile effects of testosterone are well known, yet relatively little is known about how the body compensates for a sudden loss of testosterone. Testosterone phosphorylates eNOS to generate NO through AR-dependent pathways [14]. Even though both the IPA and penis of CAST animals lose nearly half of their AR, testosterone and dihydrotestosterone-mediated relaxation is only impaired in the penis. It remains unknown why this occurs. One possibility is that estrogens derived from testosterone may contribute to IPA relaxation, but not to penile relaxation. Here, we show that a synthetic estrogen receptor beta agonist is a potent IPA vasorelaxant; however, it does not relax the penis. Another possibility may be that only a small population of AR are needed to induce vasodilation; even low doses of testosterone below physiological levels are enough to restore sexual function in testosterone deficient men [28]. There is still a possibility that testosterone-mediated IPA and penile relaxation are not dependent on AR-mediated eNOS phosphorylation.
In addition to eNOS-mediated relaxation pathways, testosterone relaxes human corpus cavernosa by activating smooth muscle adenosine triphosphate (ATP) sensitive potassium channels [29]. Testosterone hyperpolarizes vascular smooth muscle and inactivates both L-type calcium channels and voltage operated channels to cause non-endothelial-dependent relaxation [30]. Interestingly, removing the endothelial layer of Wistar Kyoto rat aortas does not impair testosterone-mediated relaxation [31]. In the current study, castration does not alter Akt and eNOS phosphorylation of testosterone incubated penises; however, there is significantly less testosterone and dihydrotestosterone-mediated CAST penile relaxation suggesting that this pathology is not of endothelial origin. To validate these findings, additional experiments confirm testosterone-mediated relaxation is not inhibited by the NO synthase inhibitor L-arginine methyl ester (data not shown). In fact, testosterone at nanomolar concentrations activates eNOS and generates NO, but pharmacological concentrations higher than 100 nanomolar induces endothelial-independent relaxation [30].
Although the direct effects of testosterone may be endothelial independent, castration still causes severe endothelial dysfunction. IPA and penises from castrated rats exhibit decreased ACh relaxation consistent with previous studies [17,32]. Endothelial dysfunction following castration is well characterized, yet the mechanisms responsible for this pathology are poorly understood. Our ex vivo vasoreactivity experiments were conducted at a physiological temperature (37°C) and to accurately understand what mechanistic changes occur after castration, IPA and penises used for molecular studies were incubated in ACh using the same myograph parameters used for ex vivo studies. At low temperatures (<20°C), eNOS maintains the dimer form; however, at 37°C, eNOS is present as both a dimer and monomer [33]. Dimeric eNOS produces NO which leads to vasorelaxation, but monomeric eNOS generates ROS leading to further endothelial damage. Our goal was to examine the interplay between endothelial NO and ROS generation and determine how it impacts vasoreactivity. At physiological temperatures, castrated IPA and penises incubated in ACh demonstrate increases in Akt and eNOS phosphorylation in conjunction with a decrease of dimeric eNOS. Further experiments are necessary to determine if increased eNOS phosphorylation may be a failed compensatory effort to increase NO production or if it may represent increased phosphorylation of monomeric eNOS. Further experiments are necessary to confirm.
Superoxide production is higher in aged hypertensive rat aortas than young hypertensive rats; inhibiting eNOS and even denuding the aortas of these rats has been shown to reduce superoxide production [20]. Although these studies demonstrate aging and castration impair endothelial function, it remains unclear why. NO is produced by dimeric eNOS, but superoxide production is the result of monomeric eNOS activation. The ratio of eNOS dimers to monomers is necessary to understand the net effect on vascular relaxation. We demonstrate Akt inhibition significantly reverses castration induced IPA and penile endothelial dysfunction. Both IPA and penis incubated in ACh contain more eNOS monomers than dimers at physiological temperatures. This suggests that ACh activation may generate a disproportionately higher level of superoxide than NO. As mentioned earlier, superoxide uncouples eNOS by depleting cofactors necessary for NO production; activating uncoupled monomeric eNOS creates a feedback loop responsible for further eNOS uncoupling [13]. Akt inhibition likely inhibits monomeric eNOS activity. In the IPA, Akt inhibition with 10DEBC preserves eNOS dimers during ACh-mediated activation. Although penile eNOS dimerization was not impacted, trace amounts of penile dimeric eNOS are evident in the CAST group. Pathological eNOS uncoupling may be too severe for drug therapy to reverse. Perhaps mitigating superoxide production will provide the best means for increasing penile endothelial-dependent relaxation.
A few limitations are present in this study. Animals are evaluated at a single time point six weeks post castration. Although animals display ED and impaired relaxation within IPA and penis, temporal changes in Akt-eNOS signaling pathways cannot be determined. Also, NO and superoxide production are not directly measured in this study. Akt inhibition causes IPA eNOS dimerization and increases ACh-mediated relaxation; however, it is unclear whether Akt inhibition specifically increases NO or reduces superoxide. A single concentration of 10DEBC was used and it is unclear if off target effects occur as well.
Conclusions
PCa survivors lack an effective ED treatment option. Akt inhibition in castrated rats demonstrates improved endothelial-dependent vasorelaxation in pre-penile and penile vasculature. Additionally, clinical trials in metastatic PCa have demonstrated a significant antitumor effect of Akt inhibitors [34]. Akt inhibitors may prove to be successful at improving erectile function safely in PCa survivors. Finally, this class of drugs may not only treat ED, but also may prevent the development of ED due to its ability to limit superoxide production.
Highlights:
Castration reduces penile and pre-penile vascular endothelial-dependent relaxation
Castration decreases the penile eNOS dimer to monomer ratio
Ex vivo Akt inhibition reverses ex vivo castration induced penile endothelial damage
Acknowledgements
Funding was provided to MRO by National Institute of Health K12-DK100024 grant and Brody School of Medicine startup funds to JLH.
Footnotes
CRediT Author Statement
MRO: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing-Original draft preparation/Review & Editing; ESP: Methodology, Validation, Investigation, Supervision, Writing – Review & Editing; JLH: Conceptualization, Methodology, Resources, Writing – Review & Editing, Supervision, Product administration, Funding acquisition.
Conflict of Interest Statement
The authors declare that there are no conflicts of interest.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
References
- [1].Gunner C, Gulamhusein A, Rosario DJ. The modern role of androgen deprivation therapy in the management of localised and locally advanced prostate cancer. J Clin Urol 2016;9:24–9. doi: 10.1177/2051415816654048. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [2].Billups KL. Erectile dysfunction as an early sign of cardiovascular disease. Int J Impot Res 2005;17:S19–24. doi: 10.1038/sj.ijir.3901425. [DOI] [PubMed] [Google Scholar]
- [3].Ng E, Corica T, Turner S, Lim A, Spry N. Sexual Function in Men with Castrate Levels of Testosterone: Observations of a Subgroup of Sexually Active Men with Prostate Cancer Undergoing Androgen Deprivation Therapy. Open J Urol 2014;04:98–103. doi: 10.4236/oju.2014.47017. [DOI] [Google Scholar]
- [4].Hoffman RM, Hunt WC, Gilliland FD, Stephenson RA, Potosky AL. Patient satisfaction with treatment decisions for clinically localized prostate carcinoma. Results from the prostate cancer outcomes study. Cancer 2003;97:1653–62. doi: 10.1002/cncr.11233. [DOI] [PubMed] [Google Scholar]
- [5].Isidori AM, Buvat J, Corona G, Goldstein I, Jannini EA, Lenzi A, et al. A critical analysis of the role of testosterone in erectile function: From pathophysiology to treatment - A systematic review. Eur Urol 2014;65:99–112. doi: 10.1016/j.eururo.2013.08.048. [DOI] [PubMed] [Google Scholar]
- [6].Keast JR, Saunders RJ. Testosterone has potent, selective effects on the morphology of pelvic autonomic neurons which control the bladder, lower bowel and internal reproductive organs of the male rat. Neuroscience 1998;85:543–56. doi: 10.1016/S0306-4522(97)00631-3. [DOI] [PubMed] [Google Scholar]
- [7].Castela A, Vendeira P, Costa C. Testosterone, Endothelial Health, and Erectile Function. ISRN Endocrinol 2011;2011:1–7. doi: 10.5402/2011/839149. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [8].Traish AM, Goldstein I, Kim NN. Testosterone and Erectile Function: From Basic Research to a New Clinical Paradigm for Managing Men with Androgen Insufficiency and Erectile Dysfunction. Eur Urol 2007;52:54–70. doi: 10.1016/j.eururo.2007.02.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [9].Tsumura H, Satoh T, Ishiyama H, Hirano S, Tabata K-I, Kurosaka S, et al. Recovery of serum testosterone following neoadjuvant and adjuvant androgen deprivation therapy in men treated with prostate brachytherapy. World J Radiol 2015;7:494–500. doi: 10.4329/wjr.v7.i12.494. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [10].Nguyen PL, Alibhai SMH, Basaria S, D’Amico A V., Kantoff PW, Keating NL, et al. Adverse effects of androgen deprivation therapy and strategies to mitigate them. Eur Urol 2015;67:825–36. doi: 10.1016/j.eururo.2014.07.010. [DOI] [PubMed] [Google Scholar]
- [11].Mobley DF, Khera M, Baum N. Recent advances in the treatment of erectile dysfunction. Postgrad Med J 2017;93:679–85. doi: 10.1136/postgradmedj-2016-134073. [DOI] [PubMed] [Google Scholar]
- [12].Hurt KJ, Musicki B, Palese MA, Crone JK, Becker RE, Moriarity JL, et al. Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection. Proc Natl Acad Sci U S A 2002;99:4061–6. doi: 10.1073/pnas.052712499. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [13].Chen CA, Druhan LJ, Varadharaj S, Chen YR, Zweier JL. Phosphorylation of endothelial nitric-oxide synthase regulates superoxide generation from the enzyme. J Biol Chem 2008;283:27038–47. doi: 10.1074/jbc.M802269200. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [14].Yu J, Akishita M, Eto M, Ogawa S, Son BK, Kato S, et al. Androgen receptor-dependent activation of endothelial nitric oxide synthase in vascular endothelial cells: Role of phosphatidylinositol 3-kinase/Akt pathway. Endocrinology 2010;151:1822–8. doi: 10.1210/en.2009-1048. [DOI] [PubMed] [Google Scholar]
- [15].Li R, Meng X, Zhang Y, Wang T, Yang J, Niu Y, et al. Testosterone improves erectile function through inhibition of reactive oxygen species generation in castrated rats. PeerJ 2016;4:e2000. doi: 10.7717/peerj.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [16].Cui K, Li R, Liu K, Wang T, Liu J, Rao K. Testosterone preserves endothelial function through regulation of S1P1/Akt/FOXO3a signalling pathway in the rat corpus cavernosum. Andrologia 2019;51:e13173. doi: 10.1111/and.13173. [DOI] [PubMed] [Google Scholar]
- [17].Kataoka T, Hotta Y, Maeda Y, Kimura K. Testosterone Deficiency Causes Endothelial Dysfunction via Elevation of Asymmetric Dimethylarginine and Oxidative Stress in Castrated Rats. J Sex Med 2017;14:1540–8. doi: 10.1016/j.jsxm.2017.11.001. [DOI] [PubMed] [Google Scholar]
- [18].Alves-Lopes R, Neves KB, Silva MA, Olivon VC, Ruginsk SG, Antunes-Rodrigues J, et al. Functional and structural changes in internal pudendal arteries underlie erectile dysfunction induced by androgen deprivation. Asian J Androl 2016:1–7. doi: 10.4103/1008-682X.173935. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [19].Shi X, Guan Y, Jiang S, Li T, Sun B, Cheng H. Renin-angiotensin system inhibitor attenuates oxidative stress induced human coronary artery endothelial cell dysfunction via the PI3K/AKT/mTOR pathway. Arch Med Sci 2019;15:152–64. doi: 10.5114/aoms.2018.74026. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [20].Hamilton CA, Brosnan MJ, McIntyre M, Graham D, Dominiczak AF. Superoxide Excess in Hypertension and Aging. Hypertension 2001;37:529–34. doi: 10.1161/01.HYP.37.2.529. [DOI] [PubMed] [Google Scholar]
- [21].Rajapakse AG, Yepuri G, Carvas JM, Stein S, Matter CM, Scerri I, et al. Hyperactive S6K1 mediates oxidative stress and endothelial dysfunction in aging: Inhibition by resveratrol. PLoS One 2011;6. doi: 10.1371/journal.pone.0019237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [22].Sinagra T, Tamburella A, Urso V, Siarkos I, Drago F, Bucolo C, et al. Reversible inhibition of vasoconstriction by thiazolidinediones related to PI3K/Akt inhibition in vascular smooth muscle cells. Biochem Pharmacol 2013;85:551–9. doi: 10.1016/j.bcp.2012.11.013. [DOI] [PubMed] [Google Scholar]
- [23].Zareba P, Duivenvoorden W, Leong DP, Pinthus JH. Androgen deprivation therapy and cardiovascular disease: what is the linking mechanism? Ther Adv Urol 2016;8:118–29. doi: 10.1177/1756287215617872. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [24].Cai JJ, Wen J, Jiang WH, Lin J, Hong Y, Zhu YS. Androgen actions on endothelium functions and cardiovascular diseases. J Geriatr Cardiol 2016;13:183–96. doi: 10.11909/j.issn.1671-5411.2016.02.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [25].Duckles SP, Miller VM. Hormonal modulation of endothelial NO production. Pflügers Arch - Eur J Physiol 2010;459:841–51. doi: 10.1007/s00424-010-0797-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [26].Alexandersen P, Haarbo J, Byrjalsen I, Lawaetz H, Christiansen C. Natural androgens inhibit male atherosclerosis: A study in castrated, cholesterol-fed rabbits. Circ Res 1999;84:813–9. doi: 10.1161/01.RES.84.7.813. [DOI] [PubMed] [Google Scholar]
- [27].Lu YL, Kuang L, Zhu H, Wu H, Wang XF, Pang YP, et al. Changes in aortic endothelium ultrastructure in male rats following castration, replacement with testosterone and administration of 5α-reductase inhibitor. Asian J Androl 2007;9:843–7. doi: 10.1111/j.1745-7262.2007.00327.x. [DOI] [PubMed] [Google Scholar]
- [28].Snyder PJ, Bhasin S, Cunningham GR, Matsumoto AM, Stephens-Shields AJ, Cauley JA, et al. Effects of Testosterone Treatment in Older Men. N Engl J Med 2016;374:611–24. doi: 10.1056/NEJMoa1506119. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [29].Yildiz O, Seyrek M, Irkilata HC, Yildirim I, Tahmaz L, Dayanc M. Testosterone Might Cause Relaxation of Human Corpus Cavernosum by Potassium Channel Opening Action. Urology 2009;74:229–32. doi: 10.1016/j.urology.2008.12.022. [DOI] [PubMed] [Google Scholar]
- [30].Perusquía M, Stallone JN. Do androgens play a beneficial role in the regulation of vascular tone? Nongenomic vascular effects of testosterone metabolites. Am J Physiol - Hear Circ Physiol 2010;298:1301–7. doi: 10.1152/ajpheart.00753.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [31].Honda H, Unemoto T, Kogo H. Different mechanisms for testosterone-induced relaxation of aorta between normotensive and spontaneously hypertensive rats. Hypertension 1999;34:1232–6. doi: 10.1161/01.HYP.34.6.1232. [DOI] [PubMed] [Google Scholar]
- [32].Rodrigues FL, Lopes RAM, Fais RS, De Oliveira L, Prado CM, Tostes RC, et al. Erectile dysfunction in heart failure rats is associated with increased neurogenic contractions in cavernous tissue and internal pudendal artery. Life Sci 2016;145:9–18. doi: 10.1016/j.lfs.2015.12.005. [DOI] [PubMed] [Google Scholar]
- [33].Venema RC, Ju H, Zou R, Ryan JW, Venema VJ. Subunit Interactions of Endothelial Nitric-oxide Synthase. J Biol Chem 2002;272:1276–82. doi: 10.1074/jbc.272.2.1276. [DOI] [PubMed] [Google Scholar]
- [34].de Bono JS, De Giorgi U, Rodrigues DN, Massard C, Bracarda S, Font A, et al. Randomized Phase II Study Evaluating Akt Blockade with Ipatasertib, in Combination with Abiraterone, in Patients with Metastatic Prostate Cancer with and without PTEN Loss. Clin Cancer Res 2019;25:928–36. doi: 10.1158/1078-0432.CCR-18-0981. [DOI] [PubMed] [Google Scholar]