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Erectile Dysfunction in Heme-Deficient Nitric Oxide–Unresponsive Soluble Guanylate Cyclase Knock-In Mice
Erectile Dysfunction in Heme-Deficient Nitric OxideeUnresponsive Soluble Guanylate Cyclase Knock-In Mice Kelly Decaluwé, PhD,1 Bart Pauwels, PhD,1 Charlotte Boydens, MSc,1 Robrecht Thoonen, PhD,2 Emmanuel S. Buys, PhD,3 Peter Brouckaert, PhD,4,5 and Johan Van de Voorde, PhD1
ABSTRACT
Introduction: The nitric oxide (NO), soluble guanylate cyclase (sGC), and cyclic guanosine monophosphate (cGMP) pathway is the leading pathway in penile erection. Aim: To assess erectile function in a mouse model in which sGC is deficient in heme (apo-sGC) and unresponsive to NO. Methods: Mutant mice (sGCb1ki/ki) that express an sGC enzyme that retains basal activity but fails to respond to NO because of heme deficiency (apo-sGC) were used. Isolated corpora cavernosa from sGCb1ki/ki and wild-type mice were mounted in vitro for isometric tension recordings in response to sGC-dependent and -independent vasorelaxant agents. In addition, the erectile effects of some of these agents were tested in vivo at intracavernosal injection. Main Outcome Measures: In vitro and in vivo recordings of erectile responses in sGCb1ki/ki and wild-type mice after stimulation with sGC-dependent and -independent vasorelaxant agents. Results: NO-induced responses were abolished in sGCb1ki/ki mice in vitro and in vivo. The ability of the hemedependent, NO-independent sGC stimulator BAY 41-2272 to relax the corpora cavernosa was markedly attenuated in sGCb1ki/ki mice. In contrast, the relaxation response to the heme- and NO-independent sGC activator BAY 58-2667 was significantly enhanced in sGCb1ki/ki mice. The relaxing effect of sGC-independent vasorelaxant agents was similar in wild-type and sGCb1ki/ki mice, illustrating that the observed alterations in vasorelaxation are limited to NO-sGC-cGMPemediated processes. Conclusion: Our results suggest that sGC is the sole target of NO in erectile physiology. Furthermore, this study provides indirect evidence that, in addition to sGCa1b1, sGCa2b1 is important for erectile function. In addition, the significant relaxation observed in sGCb1ki/ki mice with the cumulative addition of the sGC activator BAY 58-2667 indicates that sGC activators might offer value in treating erectile dysfunction. Decaluwé K, Pauwels B, Boydens C, et al. Erectile Dysfunction in Heme-Deficient Nitric Oxidee Unresponsive Soluble Guanylate Cyclase Knock-In Mice. J Sex Med 2017;14:196e204. Copyright 2016, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.
Key Words: Soluble Guanylate Cyclase; Apo-sGC; Erectile Dysfunction; Oxidative Stress; Soluble Guanylate Cyclase Activators; Soluble Guanylate Cyclase Stimulators
Received June 13, 2016. Accepted December 6, 2016. 1
Department of Pharmacology, Ghent University, Ghent, Belgium;
2
Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA;
3
Anesthesia Center for Critical Care Research, Department of Anesthesia and Critical Care and Pain Medicine, Massachusetts General Hospital Research Institute, Harvard Medical School, Boston, MA, USA;
4
5
Inflammation Research Center, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent, Belgium;
Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Copyright ª 2016, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jsxm.2016.12.007
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INTRODUCTION The discovery that the nitric oxide (NO), soluble guanylate cyclase (sGC), and cyclic guanosine monophosphate (cGMP) pathway mediates erection has led to the use of phosphodiesterase type 5 (PDE-5) inhibitors as the gold standard for treating erectile dysfunction (ED) through the inhibition of cGMP degradation.1,2 Despite their high efficacy, these drugs have limitations such as the necessity of minimum NO and cGMP formation. A substantial decrease in NO bioavailability and/or sGC sensitivity for NO causes PDE-5 inhibitor resistance.3 This situation can be expected in pathologies accompanied by high levels of oxidative stress such as obesity, diabetes, atherosclerosis, J Sex Med 2017;14:196e204
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and hypertension.4 The oxygen radicals scavenge NO and oxidize the heme group of sGC, making the enzyme unresponsive to NO.5 This leads to severe ED requiring alternative strategies that are often invasive and uncomfortable for the patient. As such, the search for new therapeutic alternatives, including sGC stimulators and activators, is needed.3,6,7 The NO receptor sGC can present advantages as a therapeutic target for ED. Recently, the therapeutic strategy by targeting sGC and cGMP has regained attention. Several studies have shown that sGC activation, irrespective of the presence of NO, induces erectile responses.3,8e13 Moreover, although sGC also is a target of oxidative damage, it can be reactivated by heme-independent sGC activators, indicating that sGC could present a valid target for ED even when associated with severe oxidative stress.14 sGC is found in two isoforms, sGCa1b1 and sGCa2b1.15 Knowledge of the functional role of these two isoforms in different tissues could lead to the development of more tissueselective therapeutics and thus minimize side effects. sGCa1b1 is the predominant isoform in the CC.16 Our previous studies describing NO-dependent erectile responses in corpora cavernosa (CC) isolated from a mouse model lacking functional sGCa1b1 (sGCa1/ mice) suggested that a mechanism other than sGCa1b1 activation must be involved in erection. Whether activation of the less abundantly expressed sGCa2b1 isoform and/or an sGC-independent mechanism might be involved remains to be determined.17,18
with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication 85-23, revised 1996). The studies were approved by the local ethical committee for animal experiments at the Faculty of Medicine and Health Sciences, Ghent University (Ghent, Belgium). On the day of the experiment, the mice were sexually mature (age ¼ 10e16 weeks).
In Vitro Study After cervical dislocation, the penile tissue was isolated and horizontally mounted on a myograph as described previously.18 The tissue chambers contained Krebs-Ringer bicarbonate (KRB) solution 10 mL at 37 C (pH 7.4) equilibrated with 95% O2 and 5% CO2. The preparations were preloaded with 0.45 g of tension and allowed to equilibrate for 60 minutes in bath fluid that was frequently replaced by fresh KRB solution. The preparations were contracted three times with norepinephrine (NOR) 5 mmol/L, washed, and allowed to relax to resting tension before starting the protocol. When the pre-contraction response reached a stable level, electrical field stimulation (EFS; train duration ¼ 20 seconds, frequency ¼ 1, 2, 4, and 8 Hz, pulse duration ¼ 5 ms, voltage ¼ 80 V), delivered by a Grass stimulator through two parallel electrodes, was applied to the tissue. In other experiments, various vasodilating substances were added at a stable tension, achieved with NOR 5 mmol/L, to analyze the relaxing response of the CC. Between response curves, the CC were washed and allowed to recover for 20 to 30 minutes.
AIMS
In Vivo Study
Recently, a new mouse model expressing apo-sGC was described as lacking NO-sensitive sGC. In these sGCb1ki/ki mice, the important histidine 105 is converted to phenylalanine in the sGCb1 subunit. This results in uncoupling of the heme group, yielding a heterodimer that retains basal cyclase activity but fails to respond to NO owing to failure of binding the heme.19 Together with studies in sGCa1/ mice, the sGCb1ki/ki mouse model allows for the differentiation of sGC-dependent and -independent physiologic pathways involved in erectile function and enables the establishment of the relative contribution of the two sGC isoforms (sGCa1b1 and sGCa2b1). In addition, this model mimics pathologic situations in which sGC is oxidized and rendered unresponsive to NO, allowing the study of the therapeutic potential of the sGC stimulator BAY 41-2272 and the sGC activator BAY 58-2667.
Mice were anesthetized with an isoflurane-oxygen breathing mixture and prepared for surgery as previously described.18 Surgical dissection was performed by exposing the left carotid artery and the CC. A polyethylene (PE)-10 tube was introduced into the left carotid artery and a 30-gauge needle attached to a PE-10 tube was inserted into the right CC. The tubes were connected to a pressure transducer and a recorder (PowerLab 4/30, ADInstruments, Oxford, UK) for the simultaneous monitoring of intracorporal pressure (ICP) and systemic mean arterial pressure (MAP). For intracavernosal drug administration, a separate cannula (30-gauge needle attached to a PE-10 tube and a 25-mL syringe) was inserted into the left corpora cavernosum. All cannulas were filled with heparinized saline (100 U/mL). Injection volume was standardized to 2 mL to ensure minimal volumerelated changes to the ICP. For electrical stimulation (ES) of the cavernous nerve, the bladder and prostate were exposed through a midline suprapubic incision and the testes and epididymis were repositioned into the abdomen after they were divided from their scrotal attachments. The bilateral cavernosal nerves were isolated lateral to the urethra, at the lower lateral portion of the prostate. An electrode with parallel hooks (0.7e1 mm), attached to a Grass S88 stimulator, was placed around the isolated nerve. The following stimulation parameters were used: 5, 10 and 15 Hz, 5ms duration, and 8 V. Each stimulation had a duration of 60 seconds and a resting interval of 15 minutes.
METHODS Animals All experiments were performed in male homozygous sGCb1ki/ki and wild-type (WT) littermates (heterozygous breeding; genetic background, mixed 129/SvJ-C57BL6/J) developed and bred in the Inflammation Research Center of the Flanders Interuniversity Institute for Biotechnology (Ghent, Belgium) as described previously.19 The animals were treated in accordance J Sex Med 2017;14:196e204
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Measurement of sGC Activity
After 30 minutes of equilibration in KRB solution at 37 C, bubbled with 95% O2 and 5% CO2 (pH ¼ 7.4), the corporal tissues were incubated with diethylamine NONOate diethyl ammonium salt (10 mmol/L) or BAY 58-2667 (10 mmol/L) or used as a control. This reaction was stopped after 1 minute of incubation by snap freezing the tissue in liquid nitrogen. Then, the collected segments were kept at 80 C until further processing. sGC enzyme activity, measured as described by Bloch et al,20 is expressed as picomoles of cGMP produced per minute per milligram of protein in CC extract supernatant.
Drugs and Chemicals The in vitro experiments were performed in KRB solution (NaCl 135 mmol/L, KCl 5 mmol/L, NaHCO3 20 mmol/L, glucose 10 mmol/L, CaCl2 2.5 mmol/L, MgSO4 1.3 mmol/L, KH2PO4 1.2 mmol/L, ethylenediaminetetra-acetic acid EDTA 0.026 mmol/L in H2O). Acetylcholine chloride (ACh), N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl]1,3-propanediamine (spermine-NO), diethylamine NONOate diethyl ammonium salt, forskolin, 8-(4-chlorophenylthio)guanosine 30 ,50 -cyclic monophosphate sodium salt (8-pCPTcGMP), and NOR bitartrate were obtained from Sigma-Aldrich (St Louis, MO, USA), BAY 41-2272 was obtained from Alexis (San Diego, CA, USA), and sodium nitroprusside (SNP) was obtained from Merck (Darmstadt, Germany). Cinaciguat (BAY 58-2667; 4-({(4-carboxybutyl)[2-(2-{[4-(2-phenylethyl)benzyl] oxy}phenyl)ethyl] amino}methyl)benzoic acid) was kindly provided by Prof Dr Johannes-Peter Stasch (Institute of Cardiovascular Research, Pharma Research Centre, Bayer Schering, Wuppertal, Germany). BAY 58-2667 was dissolved in a solution of phosphate buffered saline, diethylene glycol monomethyl ether, and Cremophor EL (60%, 20%, and 20%), BAY 41-2272 was dissolved in dimethyl sulfoxide, ACh was dissolved in potassium hydrogen phthalate buffer 50 mmol/L (pH ¼ 4.0), and forskolin was dissolved in ethanol. The other drugs were dissolved in distilled water for in vitro experiments or heparinized saline (100 U/mL) for in vivo experiments. Saturated NO solution was prepared from gas (Air Liquide, Paris, France) as described by Kelm and Schrader.21 All concentrations are expressed as final molar concentrations in the organ bath studies; for the in vivo studies, the amount of agent injected is presented as micrograms per kilogram. The final concentration of dimethyl sulfoxide or ethanol in the organ bath never surpassed 0.1%.
Figure 1. Relaxation effect of endogenous nitric oxide on pre-contracted (with norepinephrine 50 mmol/L) corpora cavernosa from sGCb1þ/þ and sGCb1ki/ki mice evoked by (A) ACh (n ¼ 8) and (B) EFS (n ¼ 8). *P < .05 for sGCb1þ/þ vs sGCb1ki/ki; # P < .01 for sGCb1þ/þ vs sGCb1ki/ki. ACh ¼ acetylcholine chloride; EFS ¼ electrical field stimulation. observations (SPSS 12, SPSS, Inc, Chicago, IL, USA). A P less than .05 was considered significant.
MAIN OUTCOME MEASURES The present study established in vitro and in vivo erectile responses in sGCb1ki/ki and WT mice with stimulation of NO to determine whether sGC-independent pathways could regulate CC relaxation. These results also allowed an evaluation of the role of the minor expressed sGCa2b1 isoform by comparing them with those obtained from sGCa1/ mice. In addition, the corporal relaxing effect of an sGC stimulator and an sGC activator in mutant and WT mice was examined. Specificity of the results was assessed by studying the erectile responses of sGC-independent activators in mutant and WT mice.
RESULTS In Vitro Results
Calculations and Statistics Data are presented as mean ± standard error of the mean; n represents the number of mice. In the in vitro studies, relaxations are expressed as the percentage of relaxation. For the in vivo study, results are calculated as ICP adjusted for MAP, expressed as the percentage (ICP/MAP 100). Statistical significance was evaluated using the Student t-test for paired and unpaired
NO- and sGC-Dependent CC Relaxation The vasorelaxant influence of endothelium-derived NO and neuronal-derived NO was examined using ACh and EFS of the intrinsic nerves, respectively, on CC isolated from WT (sGCb1þ/þ) and sGCb1ki/ki mice. ACh (10 mmol/L) relaxed NORepre-contracted CC tissues from WT mice and further contracted CC tissues from sGCb1ki/ki mice (Figure 1A). J Sex Med 2017;14:196e204
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Figure 2. Relaxation effect of exogenous NO on pre-contracted (with norepinephrine 50 mmol/L) corpora cavernosa from sGCb1þ/þ and
sGCb1ki/ki mice evoked by (A) SNP (n ¼ 8), (B) spermine-NO (n ¼ 4), and (C) NO gas (n ¼ 8). #P < .01 for sGCb1þ/þ vs sGCb1ki/ki. NO ¼ nitric oxide; SNP ¼ sodium nitroprusside; spermine-NO ¼ diethylamine NONOate diethyl ammonium salt.
Similarly, applying EFS relaxed NOR-contracted CC of WT mice in a frequency-dependent manner, an effect that was abolished in tissues of sGCb1ki/ki mice. The CC of sGCb1ki/ki even showed a tendency to contract with EFS at higher frequencies (Figure 1B). We investigated corporal responses to exogenous NO (Figure 2). All NO donors (SNP 1 nmol/L to 10 mmol/L, spermine-NO 1 nnol/L to 10 mmol/L, NO gas 1 mmol/L to 0.1 mmol/L) relaxed pre-contracted corporal tissues from WT mice in a concentrationdependent manner, a response that was significantly impaired in sGCb1ki/ki mice. It should be noted that SNP and NO gas induced mild relaxation in sGCb1ki/ki mice at higher concentrations. NO- and sGC-Independent CC Relaxation Addition of increasing concentrations of the cell membrane permeable cGMP analog, 8-pCPT-cGMP (100 nmol/L to 0.1 mmol/L) and the adenylate cyclase stimulator forskolin (1 nmol/L to 1 mmol/L), elicited identical concentration-dependent relaxations in CC preparations from WT and sGCb1ki/ki mice (Figure 3). NO-Independent but sGC-Dependent CC Relaxation We also addressed whether loss of sGC heme could affect the ability of sGC stimulators and activators to modulate corporal relaxation. Administration of BAY 41-2272 (1 nmol/L to 10 mmol/L), a NO-independent heme-dependent sGC stimulator, produced J Sex Med 2017;14:196e204
concentration-dependent relaxations in WT and sGCb1ki/ki CC tissues. In sGCb1ki/ki CC, the relaxant effect of BAY 41-2272 was attenuated (Figure 4A). Cumulative addition of the heme- and NO-independent sGC activator BAY 58-2262 (1 nmol/L to 10 mmol/L) produced concentration-dependent relaxations in WT and sGCb1ki/ki CC tissues. The relaxations were significantly potentiated in sGCb1ki/ki CC preparations (Figure 4B).
Measurement of sGC Activity The baseline activity of sGC did not differ in CC of WT and sGCb1ki/ki mice. NO increased sGC activity in the CC of WT mice but not in the CC of sGCb1ki/ki mice. Stimulation with BAY 58-2667 resulted in an upregulation of sGC activity in sGCb1þ/þ mice, but, surprisingly, this response was less pronounced in the CC of sGCb1ki/ki mice (Figure 5).
In Vivo Studies Basal ICP, when adjusted for MAP, did not differ significantly between WT and sGCb1ki/ki mice (15.7 ± 1.1% for sGCb1þ/þ [n ¼ 18] vs 11.0 ± 2.4% for sGCb1ki/ki [n ¼ 18]; P > .05). NO- and sGC-Dependent Erectile Response The in vivo effect of endogenous NO was examined by ES of the cavernosal nerve at different frequencies (8 V, 1 ms, 60 seconds, 5e15 Hz). A frequency-dependent increase in
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Figure 3. Relaxation effect of soluble guanylate cyclaseeindependent vasodilators on pre-contracted (with norepinephrine 50 mmol/L) corpora cavernosa from sGCb1þ/þ and sGCb1ki/ki mice evoked by (A) 8-pCPT-cGMP (n ¼ 5) and (B) forskolin (n ¼ 4). 8-pCPT-cGMP ¼ 8(4-chlorophenylthio)-guanosine 30 ,50 -cyclic monophosphate sodium salt.
ICP/MAP was observed in WT mice after ES, whereas ES of the cavernosal nerve in sGCb1ki/ki mice did not induce any changes in ICP/MAP (Figure 6A). Intracavernosal administration of L-nitro-arginine methyl ester before ES of the cavernosal nerve abolished the increase in ICP/MAP normally observed with ES in sGCb1þ/þ mice (unpublished observations). These data indicated that no vasodilatory substances other than NO are released during ES. Intracavernosal administration of SNP (1e4 mg/kg) and spermine-NO (10e20 mg/kg) resulted in dose-dependent increases in ICP/MAP in WT mice, but not in sGCb1ki/ki mice (Figure 6B, C). NO- and sGC-Independent Erectile Response Comparable to the in vitro results, intracavernosal administration of 8-pCPT-cGMP (5e10 mg/kg) and forskolin (1e4 mg/kg) increased the ICP/MAP equally in WT and sGCb1ki/ki mice (Figure 7).
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Figure 4. Panel A shows the relaxation effect of the nitric oxideeindependent soluble guanylate cyclase activator BAY 412272 on pre-contracted (with norepinephrine 50 mmol/L) corpora cavernosa from sGCb1þ/þ and sGCb1ki/ki mice (n ¼ 7). *P < .05 for sGCb1þ/þ vs sGCb1ki/ki. Panel B shows the relaxation effect of the nitric oxide- and heme-independent soluble guanylate cyclaseeactivator BAY 58-2667 on pre-contracted (with norepinephrine 50 mmol/L) corpora cavernosa from sGCb1þ/þ and sGCb1ki/ki mice (n ¼ 8). *P < .05 for sGCb1þ/þ vs sGCb1ki/ki.
DISCUSSION The main finding of the present study is that activation of sGC is the sole pathway leading to NO-induced erection and that the sGCa1b1 and sGCa2b1 isoforms are involved. In addition, our observations suggest that sGC activators such as
Figure 5. Soluble guanylate cyclase activity measurements in corpora cavernosa from sGCb1þ/þ and sGCb1ki/ki mice (n ¼ 6). *P < .05 for sGCb1þ/þ vs sGCb1ki/ki; #P < .05 for baseline soluble guanylate cyclase activity in sGCb1þ/þ mice vs soluble guanylate cyclase activity after addition of diethylamine NONOate diethyl ammonium salt and BAY58 in sGCb1ki/ki mice. BAY58 ¼ BAY 58-2667; NO ¼ nitric oxide; WT ¼ wild-type. J Sex Med 2017;14:196e204
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Figure 6. ICP/MAP ratio (percentage) in response to (A) ES (n ¼ 4), (B) SNP (n ¼ 5), and (C) spermine-NO (n ¼ 5). *P < .05 for sGCb1þ/þ
vs sGCb1ki/ki; #P < .01 for sGCb1þ/þ vs sGCb1ki/ki. ES ¼ electrical stimulation; ICP ¼ intracorporal pressure; MAP ¼ mean arterial pressure; SNP ¼ sodium nitroprusside; spermine-NO ¼ diethylamine NONOate diethyl ammonium salt.
BAY 58-2667 might be a therapeutic approach for patients with severe ED from increased oxidative stress as in diabetes, hypertension, atherosclerosis, and obesity. These conclusions are derived from experiments using sGCb1ki/ki mice, a model in which basal sGC activity is preserved, whereas all NO-induced sGC activation is abolished as shown by the sGC assays (Figure 5). In our previous studies examining erectile responses in sGCa1/ mice, we found that the sGCa1 subunit was required to obtain proper erectile responses to substances generally known to exert their actions by sGC.17,18 Based on our previous observation of residual relaxing responsiveness to exogenous NO in corporal tissues from sGCa1/ mice, a minor contribution of the sGCa2b1 isoform in erectile function was suggested. However, sGC-independent mechanisms could be involved. For example, activation of Ca2þ and voltage-dependent Kþ channels, sarcoplasmic reticulum Ca2þ-adenosine triphosphatase (ATPase) and Naþ/Kþ-ATPase have been described as sGC-independent vasorelaxant actions of NO in vascular smooth muscle cells.22,23 The absolute failure of NO to induce erection in sGCb1ki/ki mice shows that sGC is required for NO-induced relaxation of the CC (Figures 1, 2, and 6) and argues against a role for sGC-independent mechanisms. The observation that higher J Sex Med 2017;14:196e204
concentrations of SNP and NO gas relax CC in vitro might be due to the probability that high NO concentrations act through non-physiologic and even toxic mechanisms.24e26 These results are in line with our previous study reporting that the remaining exogenous NO-induced responses observed in sGCa1/ mice were abrogated in the presence of the sGC inhibitor 1H-(1,2,4) oxadiazolo(4,3-a)quinoxalin-1-one.17 Furthermore, our data are consistent with the findings of Groneberg et al27 who found that CC from sGC/ mice were unresponsive to NO. In addition, CC of mice lacking the cGMP target, cGMP-dependent protein kinase-1, failed to relax with activation of the NO-cGMP signaling cascade.26 Together these data support the importance of sGC as the primary and sole receptor for NO in erectile physiology and demonstrate that heme-containing reduced sGC is needed for NO effects on corporal tissues. Despite ED, aposGC mice remain fertile. This can be explained by the presence of a penis bone in mice making erection not absolutely necessary for fertilization. Epidemiologic studies have found that approximately 30% of patients with ED do not respond to PDE-5 inhibitors.8,28 Unresponsiveness to PDE-5 inhibitors might be due to the fact that NO bioavailability is impaired to such an extent that inhibition of cGMP degradation no longer has a significant therapeutic
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effect on phosphodiesterases at concentration higher than 5 mmol/L.19,31,32 Furthermore, BAY 41-2272 has been reported to induce cGMP-independent vasorelaxation through activation of Naþ-Kþ-ATPase and inhibition of Ca2þ entry.33,34 In contrast, the sGC activator BAY 58-2667 relaxed the CC in a concentration-dependent manner (Figure 4B), a response that was significantly increased in the CC of sGCb1ki/ki mice compared with WT mice. The finding that BAY 58-2667 also possessed a corporal relaxing effect in WT mice is suggestive because, even in healthy mice, a subset of the available sGC pool is free of heme and responsive to sGC activators. To our surprise, the ability of BAY 58-2667 to activate sGC was less pronounced in sGCb1ki/ki mice than in WT mice, a finding that is in contrast with our functional test results (Figure 5) and previously reported findings.19 A potential explanation for this discrepancy could be that the protein levels of at least one sGC subunit is lower in the CC of sGCb1ki/ki mice, an observation previously noted for cardiovascular tissues.19,35 Another possibility is that BAY 58-2667 exerts sGC-independent effects resulting in vascular and corporal smooth muscle relaxation.
Figure 7. ICP/MAP ratio (percentage) in response to (A) 8-pCPT-cGMP (n ¼ 4) and (B) forskolin (n ¼ 5). 8-pCPT-cGMP ¼ 8-(4-chlorophenylthio)-guanosine 30 ,50 -cyclic monophosphate sodium salt; ICP ¼ intracorporal pressure; MAP ¼ mean arterial pressure.
advantage. Furthermore, increased oxidative stress often seen in severe ED might result in the oxidation of the sGC heme group. To provide a scientific basis for the concept of targeting apo-sGC as a pharmacologic strategy for ED, erectile responses to the sGC stimulator BAY 41-2272 and the sGC activator BAY 58-2667 were examined. Previously, we reported that the relaxing effect of BAY 41-2272 was significantly decreased in the CC of sGCa1/ mice. In the present study, the ability of BAY 41-2272 to activate apo-sGC and to relax corporal tissue from sGCb1ki/ki mice was severely attenuated. Although BAY 41-2272 has been suggested to activate the two sGC isoforms, no further inhibition of the BAY 41-2272 vasodilatory response was seen in the CC isolated from sGCb1ki/ki mice (BAY 41-2272 10 mmol/L in sGCa1/ vs sGCb1ki/ki mice ¼ 17.2 ± 5.5% vs 2.5 ± 5.2%), suggesting that sGCa2b1 is the predominant target of BAY 41-2272 in the CC.29 Although BAY 41-2272 is dependent on the presence of a reduced prosthetic heme moiety of sGC, BAY 41-2272 did elicit a substantial response in the CC isolated from the sGCb1ki/ki mice (Figure 4A).30 This is in line with the observation that the sGC inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one did not completely abolish the relaxation induced by BAY 41-2272. The residual effect of BAY 41-2272 in CC from sGCb1ki/ki mice is likely due to off-target effects, including its inhibitory
Our functional data clearly illustrate the potential of BAY 58-2667 to ameliorate erectile function in mice expressing aposGC. From a clinical point of view, this finding is of major importance. Many diseases associated with ED are characterized by increased oxidative stress. This leads to lower NO bioavailability and an alteration in the redox state of sGC through oxidation of the heme iron hampering its activation by NO.36,37 For those patients, PDE-5 inhibitors will offer little therapeutic advantage in treating ED because only low concentrations of cGMP are produced. As shown by this study, sGC stimulators fail to activate oxidized sGC and thus provide no or limited advantage over PDE-5 inhibitors in restoring erectile function. The finding that BAY 58-2667 was more potent in relaxing corporal tissue expressing apo-sGC provides a solid scientific base for the further development of this new class of drugs because they might have advantages over current therapies for treating ED. This conclusion also is supported by the enhanced erectile responses seen with the administration of a different sGC activator (BAY 60-2270) after sGC oxidation and its ability to restore erectile function in obese mice and in a rat model of cavernosal nerve injury.10,12,38 Moreover, Frey et al39 presented clinical evidence that sGC activators possess erectile properties because they found that spontaneous penile erection was one of the most frequent treatment-emergent adverse events after intake of BAY 58-2667. Similar responses of CC in sGCb1þ/þ and sGCb1ki/ki mice to 8-pCPT-cGMP and forskolin showed that the pathway downstream of sGC was intact in sGCb1ki/ki mice (Figures 3 and 7). These results further illustrate that the abolished sGC-related responses are not due to an aspecific impairment of relaxation related to structural damage or to intrinsic changes in the relaxing properties of sGCb1ki/ki smooth muscle cells. Furthermore, these J Sex Med 2017;14:196e204
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data exclude a compensatory role for the adenylate cyclase and cyclic adenosine monophosphate signaling pathway.40
CONCLUSION This article describes the penile vascular phenotype of a mouse model that allowed us to distinguish between the sGC-dependent and sGC-independent effects of NO. Our data provide evidence that NO exerts its erectile properties solely through sGC activation. The present data also show the therapeutic potential of compounds able to activate apo-sGC in the setting of ED. In addition, our results suggest an important contribution of the lower expressed sGCa2b1 isoform in penile erection. This finding supports the idea of developing agents specifically targeting the sGCa2b1 isoform because this might limit potential side effects and thus provide a therapeutic advantage. The potential of sGC activators to ameliorate erectile function in several disease models and the physiologic pathways involved require further investigation.
ACKNOWLEDGMENTS We thank the animal caretakers for maintaining the animal facility. Corresponding Author: Johan Van de Voorde, PhD, Department of Pharmacology, Ghent University, De Pintelaan 185, Ghent 9000, Belgium. Tel: 32(0)9-332-3342; Fax: 32(0)9-332-8966; E-mail: [email protected] Conflicts of Interest: The authors report no conflicts of interest. Funding: Grants from FWO-Vlaanderen and the Bijzonder Onderzoeksfonds and the Geconcerteerde Onderzoeks Actie of Ghent University.
STATEMENT OF AUTHORSHIP Category 1 (a) Conception and Design Kelly Decaluwé; Johan Van de Voorde (b) Acquisition of Data Kelly Decaluwé (c) Analysis and Interpretation of Data Kelly Decaluwé; Bart Pauwels; Charlotte Boydens; Johan Van de Voorde Category 2 (a) Drafting the Article Kelly Decaluwé; Johan Van de Voorde (b) Revising It for Intellectual Content Kelly Decaluwé; Bart Pauwels; Charlotte Boydens; Robrecht Thoonen; Emmanuel S. Buys; Peter Brouckaert; Johan Van de Voorde
REFERENCES 1. Burnett AL. The role of nitric oxide in erectile dysfunction: implications for medical therapy. J Clin Hypertens (Greenwich) 2006;8:53-62. 2. Hawksworth DJ, Burnett AL. Pharmacotherapeutic management of erectile dysfunction. Clin Pharmacol Ther 2015; 98:602-610. 3. Decaluwe K, Pauwels B, Verpoest S, et al. New therapeutic targets for the treatment of erectile dysfunction. J Sex Med 2011;8:3271-3290. 4. Agarwal A, Nandipati KC, Sharma RK, et al. Role of oxidative stress in the pathophysiological mechanism of erectile dysfunction. J Androl 2006;27:335-347. 5. Hoffmann LS, Schmidt PM, Keim Y, et al. Fluorescence dequenching makes haem-free soluble guanylate cyclase detectable in living cells. PLoS One 2011;6:e23596. 6. Eardley I, Donatucci C, Corbin J, et al. Pharmacotherapy for erectile dysfunction. J Sex Med 2010;7:524-540. 7. Peak TC, Yafi FA, Sangkum P, et al. Emerging drugs for the treatment of erectile dysfunction. Expert Opin Emerg Drugs 2015;20:263-275. 8. Gur S, Kadowitz PJ, Hellstrom WJG. Exploring the potential of NO-independent stimulators and activators of soluble guanylate cyclase for the medical treatment of erectile dysfunction. Curr Pharm Des 2010;16:1619-1633. 9. Decaluwe K, Pauwels B, Boydens C, et al. Divergent molecular mechanisms underlay CO- and CORM-2einduced relaxation of corpora cavernosa. J Sex Med 2012;9:2284-2292. 10. Lasker GF, Pankey EA, Frink TJ, et al. The sGC activator BAY 60-2770 has potent erectile activity in the rat. Am J Physiol Heart Circ Physiol 2013;304:H1670-H1679. 11. Pauwels B, Boydens C, Brouckaert P, et al. Oximes induce erection and are resistant to oxidative stress. J Sex Med 2015; 12:906-915. 12. Estancial CS, Rodrigues RL, De Nucci G, et al. Pharmacological characterisation of the relaxation induced by the soluble guanylate cyclase activator, BAY 60-2770 in rabbit corpus cavernosum. BJU Int 2015;116:657-664. 13. Nunes KP, Teixeira CE, Priviero FBM, et al. Beneficial effect of the soluble guanylyl cyclase stimulator BAY 41-2272 on impaired penile erection in db/db(/) type II diabetic and obese mice. J Pharmacol Exp Ther 2015;353:330-339. 14. Follmann M, Griebenow N, Hahn MG, et al. The chemistry and biology of soluble guanylate cyclase stimulators and activators. Angew Chem Int Edit Engl 2013;52:9442-9462. 15. Koesling D, Russwurm M, Mergia E, et al. Nitric oxideesensitive guanylyl cyclase: structure and regulation. Neurochem Int 2004;45:813-819.
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16. Behrends S, Steenpass A, Porst H, et al. Expression of nitric oxide-sensitive guanylyl cyclase subunits in human corpus cavernosum. Biochem Pharmacol 2000;59:713-717.
(a) Final Approval of the Completed Article Kelly Decaluwé; Bart Pauwels; Charlotte Boydens; Robrecht Thoonen; Emmanuel S. Buys; Peter Brouckaert; Johan Van de Voorde
17. Nimmegeers S, Sips P, Buys E, et al. Role of the soluble guanylyl cyclase alpha(1)-subunit in mice corpus cavernosum smooth muscle relaxation. Int J Impot Res 2008; 20:278-284.
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204 18. Decaluwe K, Nimmegeers S, Thoonen R, et al. In vitro and in vivo studies on the importance of the soluble guanylyl cyclase alpha(1) subunit in penile erection. World J Urol 2010; 28:643-650. 19. Thoonen R, Cauwels A, Decaluwe K, et al. Cardiovascular and pharmacological implications of haem-deficient NOunresponsive soluble guanylate cyclase knock-in mice. Nat Commun 2015;6:8482. 20. Bloch KD, Filippov G, Sanchez LS, et al. Pulmonary soluble guanylate cyclase, a nitric oxide receptor, is increased during the perinatal period. Am J Physiol Lung Cell Mol Physiol 1997;272:L400-L406.
Decaluwé et al 30. Stasch JP, Hobbs AJ. NO-independent, haem-dependent soluble guanylate cyclase stimulators. Handb Exp Pharmacol 2009:277-308. 31. Stasch JP, Becker EM, Alonso-Alija C, et al. NO-independent regulatory site on soluble guanylate cyclase. Nature 2001; 410:212-215. 32. Martin E, Lee YC, Murad F. YC-1 activation of human soluble guanylyl cyclase has both heme-dependent and hemeindependent components. Proc Natl Acad Sci U S A 2001; 98:12938-12942.
21. Kelm M, Schrader J. Control of coronary vascular tone by nitric-oxide. Circ Res 1990;66:1561-1575.
33. Teixeira CE, Priviero FB, Todd J Jr, et al. Vasorelaxing effect of BAY 41-2272 in rat basilar artery: involvement of cGMPdependent and independent mechanisms. Hypertension 2006;47:596-602.
22. Gupta S, Moreland RB, Munarriz R, et al. Possible role of Naþ-Kþ-ATPase in the regulation of human corpus cavernosum smooth-muscle contractility by nitric-oxide. Br J Pharmacol 1995;116:2201-2206.
34. Miyaoka R, Mendes C, Schenka A, et al. BAY 41-2272, a soluble guanylate cyclase stimulator, relaxes isolated human ureter in a standardized in vitro model. Urology 2014;83:256; e1-7.
23. Mistry DK, Garland CJ. Nitric oxide (NO)-induced activation of large conductance Ca2þ-dependent Kf channels (BKCa) in smooth muscle cells isolated from the rat mesenteric artery. Br J Pharmacol 1998;124:1131-1140.
35. Meurer S, Pioch S, Pabst T, et al. Nitric oxideeindependent vasodilator rescues heme-oxidized soluble guanylate cyclase from proteosomal degradation. Circ Res 2009;105:33-41.
24. Friebe A, Koesling D. The function of NO-sensitive guanylyl cyclase: what we can learn from genetic mouse models. Nitric Oxide 2009;21:149-156. 25. Roncaroli F, van Eldik R, Olabe JA. Release of NO from reduced nitroprusside ion. Iron-dinitrosyl formation and NO-disproportionation reactions. Inorg Chem 2005; 44:2781-2790. 26. Hedlund P, Aszodi A, Pfeifer A, et al. Erectile dysfunction in cyclic GMP-dependent kinase Iedeficient mice. Proc Natl Acad Sci U S A 2000;97:2349-2354. 27. Groneberg D, Lies B, Konig P, et al. Preserved fertility despite erectile dysfunction in mice lacking the nitric oxide receptor. J Physiol 2013;591:491-502.
36. Sharina IG, Martin E. The role of reactive oxygen and nitrogen species in the expression and splicing of nitric oxide receptor. Antioxid Redox Signal doi:10.1089/ars.2016.6687. E-pub ahead of print. 37. Silva FH, Lanaro C, Leiria LO, et al. Oxidative stress associated with middle aging leads to sympathetic hyperactivity and downregulation of soluble guanylyl cyclase in corpus cavernosum. Am J Physiol Heart Circ Physiol 2014; 307:H1393-1400. 38. Silva FH, Leiria LO, Alexandre EC, et al. Prolonged therapy with the soluble guanylyl cyclase activator BAY 60-2770 restores the erectile function in obese mice. J Sex Med 2014; 11:2661-2670.
28. Albersen M, Shindel AW, Mwamukonda KB, et al. The future is today: emerging drugs for the treatment of erectile dysfunction. Expert Opin Emerg Drugs 2010;15:467-480.
39. Frey R, Scheerans C, Blunck M, et al. Pharmacokinetics of the soluble guanylate cyclase activator cinaciguat in individuals with hepatic impairment. J Clin Pharmacol 2012; 52:1714-1724.
29. Koglin M, Stasch JP, Behrends S. BAY 41-2272 activates two isoforms of nitric oxide-sensitive guanylyl cyclase. Biochem Biophys Res Commun 2002;292:1057-1062.
40. Uckert S, Hedlund P, Waldkirch E, et al. Interactions between cGMP- and cAMP-pathways are involved in the regulation of penile smooth muscle tone. World J Urol 2004;22:261-266.
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ABSTRACT
Introduction: The nitric oxide (NO), soluble guanylate cyclase (sGC), and cyclic guanosine monophosphate (cGMP) pathway is the leading pathway in penile erection. Aim: To assess erectile function in a mouse model in which sGC is deficient in heme (apo-sGC) and unresponsive to NO. Methods: Mutant mice (sGCb1ki/ki) that express an sGC enzyme that retains basal activity but fails to respond to NO because of heme deficiency (apo-sGC) were used. Isolated corpora cavernosa from sGCb1ki/ki and wild-type mice were mounted in vitro for isometric tension recordings in response to sGC-dependent and -independent vasorelaxant agents. In addition, the erectile effects of some of these agents were tested in vivo at intracavernosal injection. Main Outcome Measures: In vitro and in vivo recordings of erectile responses in sGCb1ki/ki and wild-type mice after stimulation with sGC-dependent and -independent vasorelaxant agents. Results: NO-induced responses were abolished in sGCb1ki/ki mice in vitro and in vivo. The ability of the hemedependent, NO-independent sGC stimulator BAY 41-2272 to relax the corpora cavernosa was markedly attenuated in sGCb1ki/ki mice. In contrast, the relaxation response to the heme- and NO-independent sGC activator BAY 58-2667 was significantly enhanced in sGCb1ki/ki mice. The relaxing effect of sGC-independent vasorelaxant agents was similar in wild-type and sGCb1ki/ki mice, illustrating that the observed alterations in vasorelaxation are limited to NO-sGC-cGMPemediated processes. Conclusion: Our results suggest that sGC is the sole target of NO in erectile physiology. Furthermore, this study provides indirect evidence that, in addition to sGCa1b1, sGCa2b1 is important for erectile function. In addition, the significant relaxation observed in sGCb1ki/ki mice with the cumulative addition of the sGC activator BAY 58-2667 indicates that sGC activators might offer value in treating erectile dysfunction. Decaluwé K, Pauwels B, Boydens C, et al. Erectile Dysfunction in Heme-Deficient Nitric Oxidee Unresponsive Soluble Guanylate Cyclase Knock-In Mice. J Sex Med 2017;14:196e204. Copyright 2016, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.
Key Words: Soluble Guanylate Cyclase; Apo-sGC; Erectile Dysfunction; Oxidative Stress; Soluble Guanylate Cyclase Activators; Soluble Guanylate Cyclase Stimulators
Received June 13, 2016. Accepted December 6, 2016. 1
Department of Pharmacology, Ghent University, Ghent, Belgium;
2
Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA;
3
Anesthesia Center for Critical Care Research, Department of Anesthesia and Critical Care and Pain Medicine, Massachusetts General Hospital Research Institute, Harvard Medical School, Boston, MA, USA;
4
5
Inflammation Research Center, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent, Belgium;
Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Copyright ª 2016, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jsxm.2016.12.007
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INTRODUCTION The discovery that the nitric oxide (NO), soluble guanylate cyclase (sGC), and cyclic guanosine monophosphate (cGMP) pathway mediates erection has led to the use of phosphodiesterase type 5 (PDE-5) inhibitors as the gold standard for treating erectile dysfunction (ED) through the inhibition of cGMP degradation.1,2 Despite their high efficacy, these drugs have limitations such as the necessity of minimum NO and cGMP formation. A substantial decrease in NO bioavailability and/or sGC sensitivity for NO causes PDE-5 inhibitor resistance.3 This situation can be expected in pathologies accompanied by high levels of oxidative stress such as obesity, diabetes, atherosclerosis, J Sex Med 2017;14:196e204
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and hypertension.4 The oxygen radicals scavenge NO and oxidize the heme group of sGC, making the enzyme unresponsive to NO.5 This leads to severe ED requiring alternative strategies that are often invasive and uncomfortable for the patient. As such, the search for new therapeutic alternatives, including sGC stimulators and activators, is needed.3,6,7 The NO receptor sGC can present advantages as a therapeutic target for ED. Recently, the therapeutic strategy by targeting sGC and cGMP has regained attention. Several studies have shown that sGC activation, irrespective of the presence of NO, induces erectile responses.3,8e13 Moreover, although sGC also is a target of oxidative damage, it can be reactivated by heme-independent sGC activators, indicating that sGC could present a valid target for ED even when associated with severe oxidative stress.14 sGC is found in two isoforms, sGCa1b1 and sGCa2b1.15 Knowledge of the functional role of these two isoforms in different tissues could lead to the development of more tissueselective therapeutics and thus minimize side effects. sGCa1b1 is the predominant isoform in the CC.16 Our previous studies describing NO-dependent erectile responses in corpora cavernosa (CC) isolated from a mouse model lacking functional sGCa1b1 (sGCa1/ mice) suggested that a mechanism other than sGCa1b1 activation must be involved in erection. Whether activation of the less abundantly expressed sGCa2b1 isoform and/or an sGC-independent mechanism might be involved remains to be determined.17,18
with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication 85-23, revised 1996). The studies were approved by the local ethical committee for animal experiments at the Faculty of Medicine and Health Sciences, Ghent University (Ghent, Belgium). On the day of the experiment, the mice were sexually mature (age ¼ 10e16 weeks).
In Vitro Study After cervical dislocation, the penile tissue was isolated and horizontally mounted on a myograph as described previously.18 The tissue chambers contained Krebs-Ringer bicarbonate (KRB) solution 10 mL at 37 C (pH 7.4) equilibrated with 95% O2 and 5% CO2. The preparations were preloaded with 0.45 g of tension and allowed to equilibrate for 60 minutes in bath fluid that was frequently replaced by fresh KRB solution. The preparations were contracted three times with norepinephrine (NOR) 5 mmol/L, washed, and allowed to relax to resting tension before starting the protocol. When the pre-contraction response reached a stable level, electrical field stimulation (EFS; train duration ¼ 20 seconds, frequency ¼ 1, 2, 4, and 8 Hz, pulse duration ¼ 5 ms, voltage ¼ 80 V), delivered by a Grass stimulator through two parallel electrodes, was applied to the tissue. In other experiments, various vasodilating substances were added at a stable tension, achieved with NOR 5 mmol/L, to analyze the relaxing response of the CC. Between response curves, the CC were washed and allowed to recover for 20 to 30 minutes.
AIMS
In Vivo Study
Recently, a new mouse model expressing apo-sGC was described as lacking NO-sensitive sGC. In these sGCb1ki/ki mice, the important histidine 105 is converted to phenylalanine in the sGCb1 subunit. This results in uncoupling of the heme group, yielding a heterodimer that retains basal cyclase activity but fails to respond to NO owing to failure of binding the heme.19 Together with studies in sGCa1/ mice, the sGCb1ki/ki mouse model allows for the differentiation of sGC-dependent and -independent physiologic pathways involved in erectile function and enables the establishment of the relative contribution of the two sGC isoforms (sGCa1b1 and sGCa2b1). In addition, this model mimics pathologic situations in which sGC is oxidized and rendered unresponsive to NO, allowing the study of the therapeutic potential of the sGC stimulator BAY 41-2272 and the sGC activator BAY 58-2667.
Mice were anesthetized with an isoflurane-oxygen breathing mixture and prepared for surgery as previously described.18 Surgical dissection was performed by exposing the left carotid artery and the CC. A polyethylene (PE)-10 tube was introduced into the left carotid artery and a 30-gauge needle attached to a PE-10 tube was inserted into the right CC. The tubes were connected to a pressure transducer and a recorder (PowerLab 4/30, ADInstruments, Oxford, UK) for the simultaneous monitoring of intracorporal pressure (ICP) and systemic mean arterial pressure (MAP). For intracavernosal drug administration, a separate cannula (30-gauge needle attached to a PE-10 tube and a 25-mL syringe) was inserted into the left corpora cavernosum. All cannulas were filled with heparinized saline (100 U/mL). Injection volume was standardized to 2 mL to ensure minimal volumerelated changes to the ICP. For electrical stimulation (ES) of the cavernous nerve, the bladder and prostate were exposed through a midline suprapubic incision and the testes and epididymis were repositioned into the abdomen after they were divided from their scrotal attachments. The bilateral cavernosal nerves were isolated lateral to the urethra, at the lower lateral portion of the prostate. An electrode with parallel hooks (0.7e1 mm), attached to a Grass S88 stimulator, was placed around the isolated nerve. The following stimulation parameters were used: 5, 10 and 15 Hz, 5ms duration, and 8 V. Each stimulation had a duration of 60 seconds and a resting interval of 15 minutes.
METHODS Animals All experiments were performed in male homozygous sGCb1ki/ki and wild-type (WT) littermates (heterozygous breeding; genetic background, mixed 129/SvJ-C57BL6/J) developed and bred in the Inflammation Research Center of the Flanders Interuniversity Institute for Biotechnology (Ghent, Belgium) as described previously.19 The animals were treated in accordance J Sex Med 2017;14:196e204
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Measurement of sGC Activity
After 30 minutes of equilibration in KRB solution at 37 C, bubbled with 95% O2 and 5% CO2 (pH ¼ 7.4), the corporal tissues were incubated with diethylamine NONOate diethyl ammonium salt (10 mmol/L) or BAY 58-2667 (10 mmol/L) or used as a control. This reaction was stopped after 1 minute of incubation by snap freezing the tissue in liquid nitrogen. Then, the collected segments were kept at 80 C until further processing. sGC enzyme activity, measured as described by Bloch et al,20 is expressed as picomoles of cGMP produced per minute per milligram of protein in CC extract supernatant.
Drugs and Chemicals The in vitro experiments were performed in KRB solution (NaCl 135 mmol/L, KCl 5 mmol/L, NaHCO3 20 mmol/L, glucose 10 mmol/L, CaCl2 2.5 mmol/L, MgSO4 1.3 mmol/L, KH2PO4 1.2 mmol/L, ethylenediaminetetra-acetic acid EDTA 0.026 mmol/L in H2O). Acetylcholine chloride (ACh), N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl]1,3-propanediamine (spermine-NO), diethylamine NONOate diethyl ammonium salt, forskolin, 8-(4-chlorophenylthio)guanosine 30 ,50 -cyclic monophosphate sodium salt (8-pCPTcGMP), and NOR bitartrate were obtained from Sigma-Aldrich (St Louis, MO, USA), BAY 41-2272 was obtained from Alexis (San Diego, CA, USA), and sodium nitroprusside (SNP) was obtained from Merck (Darmstadt, Germany). Cinaciguat (BAY 58-2667; 4-({(4-carboxybutyl)[2-(2-{[4-(2-phenylethyl)benzyl] oxy}phenyl)ethyl] amino}methyl)benzoic acid) was kindly provided by Prof Dr Johannes-Peter Stasch (Institute of Cardiovascular Research, Pharma Research Centre, Bayer Schering, Wuppertal, Germany). BAY 58-2667 was dissolved in a solution of phosphate buffered saline, diethylene glycol monomethyl ether, and Cremophor EL (60%, 20%, and 20%), BAY 41-2272 was dissolved in dimethyl sulfoxide, ACh was dissolved in potassium hydrogen phthalate buffer 50 mmol/L (pH ¼ 4.0), and forskolin was dissolved in ethanol. The other drugs were dissolved in distilled water for in vitro experiments or heparinized saline (100 U/mL) for in vivo experiments. Saturated NO solution was prepared from gas (Air Liquide, Paris, France) as described by Kelm and Schrader.21 All concentrations are expressed as final molar concentrations in the organ bath studies; for the in vivo studies, the amount of agent injected is presented as micrograms per kilogram. The final concentration of dimethyl sulfoxide or ethanol in the organ bath never surpassed 0.1%.
Figure 1. Relaxation effect of endogenous nitric oxide on pre-contracted (with norepinephrine 50 mmol/L) corpora cavernosa from sGCb1þ/þ and sGCb1ki/ki mice evoked by (A) ACh (n ¼ 8) and (B) EFS (n ¼ 8). *P < .05 for sGCb1þ/þ vs sGCb1ki/ki; # P < .01 for sGCb1þ/þ vs sGCb1ki/ki. ACh ¼ acetylcholine chloride; EFS ¼ electrical field stimulation. observations (SPSS 12, SPSS, Inc, Chicago, IL, USA). A P less than .05 was considered significant.
MAIN OUTCOME MEASURES The present study established in vitro and in vivo erectile responses in sGCb1ki/ki and WT mice with stimulation of NO to determine whether sGC-independent pathways could regulate CC relaxation. These results also allowed an evaluation of the role of the minor expressed sGCa2b1 isoform by comparing them with those obtained from sGCa1/ mice. In addition, the corporal relaxing effect of an sGC stimulator and an sGC activator in mutant and WT mice was examined. Specificity of the results was assessed by studying the erectile responses of sGC-independent activators in mutant and WT mice.
RESULTS In Vitro Results
Calculations and Statistics Data are presented as mean ± standard error of the mean; n represents the number of mice. In the in vitro studies, relaxations are expressed as the percentage of relaxation. For the in vivo study, results are calculated as ICP adjusted for MAP, expressed as the percentage (ICP/MAP 100). Statistical significance was evaluated using the Student t-test for paired and unpaired
NO- and sGC-Dependent CC Relaxation The vasorelaxant influence of endothelium-derived NO and neuronal-derived NO was examined using ACh and EFS of the intrinsic nerves, respectively, on CC isolated from WT (sGCb1þ/þ) and sGCb1ki/ki mice. ACh (10 mmol/L) relaxed NORepre-contracted CC tissues from WT mice and further contracted CC tissues from sGCb1ki/ki mice (Figure 1A). J Sex Med 2017;14:196e204
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Figure 2. Relaxation effect of exogenous NO on pre-contracted (with norepinephrine 50 mmol/L) corpora cavernosa from sGCb1þ/þ and
sGCb1ki/ki mice evoked by (A) SNP (n ¼ 8), (B) spermine-NO (n ¼ 4), and (C) NO gas (n ¼ 8). #P < .01 for sGCb1þ/þ vs sGCb1ki/ki. NO ¼ nitric oxide; SNP ¼ sodium nitroprusside; spermine-NO ¼ diethylamine NONOate diethyl ammonium salt.
Similarly, applying EFS relaxed NOR-contracted CC of WT mice in a frequency-dependent manner, an effect that was abolished in tissues of sGCb1ki/ki mice. The CC of sGCb1ki/ki even showed a tendency to contract with EFS at higher frequencies (Figure 1B). We investigated corporal responses to exogenous NO (Figure 2). All NO donors (SNP 1 nmol/L to 10 mmol/L, spermine-NO 1 nnol/L to 10 mmol/L, NO gas 1 mmol/L to 0.1 mmol/L) relaxed pre-contracted corporal tissues from WT mice in a concentrationdependent manner, a response that was significantly impaired in sGCb1ki/ki mice. It should be noted that SNP and NO gas induced mild relaxation in sGCb1ki/ki mice at higher concentrations. NO- and sGC-Independent CC Relaxation Addition of increasing concentrations of the cell membrane permeable cGMP analog, 8-pCPT-cGMP (100 nmol/L to 0.1 mmol/L) and the adenylate cyclase stimulator forskolin (1 nmol/L to 1 mmol/L), elicited identical concentration-dependent relaxations in CC preparations from WT and sGCb1ki/ki mice (Figure 3). NO-Independent but sGC-Dependent CC Relaxation We also addressed whether loss of sGC heme could affect the ability of sGC stimulators and activators to modulate corporal relaxation. Administration of BAY 41-2272 (1 nmol/L to 10 mmol/L), a NO-independent heme-dependent sGC stimulator, produced J Sex Med 2017;14:196e204
concentration-dependent relaxations in WT and sGCb1ki/ki CC tissues. In sGCb1ki/ki CC, the relaxant effect of BAY 41-2272 was attenuated (Figure 4A). Cumulative addition of the heme- and NO-independent sGC activator BAY 58-2262 (1 nmol/L to 10 mmol/L) produced concentration-dependent relaxations in WT and sGCb1ki/ki CC tissues. The relaxations were significantly potentiated in sGCb1ki/ki CC preparations (Figure 4B).
Measurement of sGC Activity The baseline activity of sGC did not differ in CC of WT and sGCb1ki/ki mice. NO increased sGC activity in the CC of WT mice but not in the CC of sGCb1ki/ki mice. Stimulation with BAY 58-2667 resulted in an upregulation of sGC activity in sGCb1þ/þ mice, but, surprisingly, this response was less pronounced in the CC of sGCb1ki/ki mice (Figure 5).
In Vivo Studies Basal ICP, when adjusted for MAP, did not differ significantly between WT and sGCb1ki/ki mice (15.7 ± 1.1% for sGCb1þ/þ [n ¼ 18] vs 11.0 ± 2.4% for sGCb1ki/ki [n ¼ 18]; P > .05). NO- and sGC-Dependent Erectile Response The in vivo effect of endogenous NO was examined by ES of the cavernosal nerve at different frequencies (8 V, 1 ms, 60 seconds, 5e15 Hz). A frequency-dependent increase in
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Figure 3. Relaxation effect of soluble guanylate cyclaseeindependent vasodilators on pre-contracted (with norepinephrine 50 mmol/L) corpora cavernosa from sGCb1þ/þ and sGCb1ki/ki mice evoked by (A) 8-pCPT-cGMP (n ¼ 5) and (B) forskolin (n ¼ 4). 8-pCPT-cGMP ¼ 8(4-chlorophenylthio)-guanosine 30 ,50 -cyclic monophosphate sodium salt.
ICP/MAP was observed in WT mice after ES, whereas ES of the cavernosal nerve in sGCb1ki/ki mice did not induce any changes in ICP/MAP (Figure 6A). Intracavernosal administration of L-nitro-arginine methyl ester before ES of the cavernosal nerve abolished the increase in ICP/MAP normally observed with ES in sGCb1þ/þ mice (unpublished observations). These data indicated that no vasodilatory substances other than NO are released during ES. Intracavernosal administration of SNP (1e4 mg/kg) and spermine-NO (10e20 mg/kg) resulted in dose-dependent increases in ICP/MAP in WT mice, but not in sGCb1ki/ki mice (Figure 6B, C). NO- and sGC-Independent Erectile Response Comparable to the in vitro results, intracavernosal administration of 8-pCPT-cGMP (5e10 mg/kg) and forskolin (1e4 mg/kg) increased the ICP/MAP equally in WT and sGCb1ki/ki mice (Figure 7).
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Figure 4. Panel A shows the relaxation effect of the nitric oxideeindependent soluble guanylate cyclase activator BAY 412272 on pre-contracted (with norepinephrine 50 mmol/L) corpora cavernosa from sGCb1þ/þ and sGCb1ki/ki mice (n ¼ 7). *P < .05 for sGCb1þ/þ vs sGCb1ki/ki. Panel B shows the relaxation effect of the nitric oxide- and heme-independent soluble guanylate cyclaseeactivator BAY 58-2667 on pre-contracted (with norepinephrine 50 mmol/L) corpora cavernosa from sGCb1þ/þ and sGCb1ki/ki mice (n ¼ 8). *P < .05 for sGCb1þ/þ vs sGCb1ki/ki.
DISCUSSION The main finding of the present study is that activation of sGC is the sole pathway leading to NO-induced erection and that the sGCa1b1 and sGCa2b1 isoforms are involved. In addition, our observations suggest that sGC activators such as
Figure 5. Soluble guanylate cyclase activity measurements in corpora cavernosa from sGCb1þ/þ and sGCb1ki/ki mice (n ¼ 6). *P < .05 for sGCb1þ/þ vs sGCb1ki/ki; #P < .05 for baseline soluble guanylate cyclase activity in sGCb1þ/þ mice vs soluble guanylate cyclase activity after addition of diethylamine NONOate diethyl ammonium salt and BAY58 in sGCb1ki/ki mice. BAY58 ¼ BAY 58-2667; NO ¼ nitric oxide; WT ¼ wild-type. J Sex Med 2017;14:196e204
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Figure 6. ICP/MAP ratio (percentage) in response to (A) ES (n ¼ 4), (B) SNP (n ¼ 5), and (C) spermine-NO (n ¼ 5). *P < .05 for sGCb1þ/þ
vs sGCb1ki/ki; #P < .01 for sGCb1þ/þ vs sGCb1ki/ki. ES ¼ electrical stimulation; ICP ¼ intracorporal pressure; MAP ¼ mean arterial pressure; SNP ¼ sodium nitroprusside; spermine-NO ¼ diethylamine NONOate diethyl ammonium salt.
BAY 58-2667 might be a therapeutic approach for patients with severe ED from increased oxidative stress as in diabetes, hypertension, atherosclerosis, and obesity. These conclusions are derived from experiments using sGCb1ki/ki mice, a model in which basal sGC activity is preserved, whereas all NO-induced sGC activation is abolished as shown by the sGC assays (Figure 5). In our previous studies examining erectile responses in sGCa1/ mice, we found that the sGCa1 subunit was required to obtain proper erectile responses to substances generally known to exert their actions by sGC.17,18 Based on our previous observation of residual relaxing responsiveness to exogenous NO in corporal tissues from sGCa1/ mice, a minor contribution of the sGCa2b1 isoform in erectile function was suggested. However, sGC-independent mechanisms could be involved. For example, activation of Ca2þ and voltage-dependent Kþ channels, sarcoplasmic reticulum Ca2þ-adenosine triphosphatase (ATPase) and Naþ/Kþ-ATPase have been described as sGC-independent vasorelaxant actions of NO in vascular smooth muscle cells.22,23 The absolute failure of NO to induce erection in sGCb1ki/ki mice shows that sGC is required for NO-induced relaxation of the CC (Figures 1, 2, and 6) and argues against a role for sGC-independent mechanisms. The observation that higher J Sex Med 2017;14:196e204
concentrations of SNP and NO gas relax CC in vitro might be due to the probability that high NO concentrations act through non-physiologic and even toxic mechanisms.24e26 These results are in line with our previous study reporting that the remaining exogenous NO-induced responses observed in sGCa1/ mice were abrogated in the presence of the sGC inhibitor 1H-(1,2,4) oxadiazolo(4,3-a)quinoxalin-1-one.17 Furthermore, our data are consistent with the findings of Groneberg et al27 who found that CC from sGC/ mice were unresponsive to NO. In addition, CC of mice lacking the cGMP target, cGMP-dependent protein kinase-1, failed to relax with activation of the NO-cGMP signaling cascade.26 Together these data support the importance of sGC as the primary and sole receptor for NO in erectile physiology and demonstrate that heme-containing reduced sGC is needed for NO effects on corporal tissues. Despite ED, aposGC mice remain fertile. This can be explained by the presence of a penis bone in mice making erection not absolutely necessary for fertilization. Epidemiologic studies have found that approximately 30% of patients with ED do not respond to PDE-5 inhibitors.8,28 Unresponsiveness to PDE-5 inhibitors might be due to the fact that NO bioavailability is impaired to such an extent that inhibition of cGMP degradation no longer has a significant therapeutic
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effect on phosphodiesterases at concentration higher than 5 mmol/L.19,31,32 Furthermore, BAY 41-2272 has been reported to induce cGMP-independent vasorelaxation through activation of Naþ-Kþ-ATPase and inhibition of Ca2þ entry.33,34 In contrast, the sGC activator BAY 58-2667 relaxed the CC in a concentration-dependent manner (Figure 4B), a response that was significantly increased in the CC of sGCb1ki/ki mice compared with WT mice. The finding that BAY 58-2667 also possessed a corporal relaxing effect in WT mice is suggestive because, even in healthy mice, a subset of the available sGC pool is free of heme and responsive to sGC activators. To our surprise, the ability of BAY 58-2667 to activate sGC was less pronounced in sGCb1ki/ki mice than in WT mice, a finding that is in contrast with our functional test results (Figure 5) and previously reported findings.19 A potential explanation for this discrepancy could be that the protein levels of at least one sGC subunit is lower in the CC of sGCb1ki/ki mice, an observation previously noted for cardiovascular tissues.19,35 Another possibility is that BAY 58-2667 exerts sGC-independent effects resulting in vascular and corporal smooth muscle relaxation.
Figure 7. ICP/MAP ratio (percentage) in response to (A) 8-pCPT-cGMP (n ¼ 4) and (B) forskolin (n ¼ 5). 8-pCPT-cGMP ¼ 8-(4-chlorophenylthio)-guanosine 30 ,50 -cyclic monophosphate sodium salt; ICP ¼ intracorporal pressure; MAP ¼ mean arterial pressure.
advantage. Furthermore, increased oxidative stress often seen in severe ED might result in the oxidation of the sGC heme group. To provide a scientific basis for the concept of targeting apo-sGC as a pharmacologic strategy for ED, erectile responses to the sGC stimulator BAY 41-2272 and the sGC activator BAY 58-2667 were examined. Previously, we reported that the relaxing effect of BAY 41-2272 was significantly decreased in the CC of sGCa1/ mice. In the present study, the ability of BAY 41-2272 to activate apo-sGC and to relax corporal tissue from sGCb1ki/ki mice was severely attenuated. Although BAY 41-2272 has been suggested to activate the two sGC isoforms, no further inhibition of the BAY 41-2272 vasodilatory response was seen in the CC isolated from sGCb1ki/ki mice (BAY 41-2272 10 mmol/L in sGCa1/ vs sGCb1ki/ki mice ¼ 17.2 ± 5.5% vs 2.5 ± 5.2%), suggesting that sGCa2b1 is the predominant target of BAY 41-2272 in the CC.29 Although BAY 41-2272 is dependent on the presence of a reduced prosthetic heme moiety of sGC, BAY 41-2272 did elicit a substantial response in the CC isolated from the sGCb1ki/ki mice (Figure 4A).30 This is in line with the observation that the sGC inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one did not completely abolish the relaxation induced by BAY 41-2272. The residual effect of BAY 41-2272 in CC from sGCb1ki/ki mice is likely due to off-target effects, including its inhibitory
Our functional data clearly illustrate the potential of BAY 58-2667 to ameliorate erectile function in mice expressing aposGC. From a clinical point of view, this finding is of major importance. Many diseases associated with ED are characterized by increased oxidative stress. This leads to lower NO bioavailability and an alteration in the redox state of sGC through oxidation of the heme iron hampering its activation by NO.36,37 For those patients, PDE-5 inhibitors will offer little therapeutic advantage in treating ED because only low concentrations of cGMP are produced. As shown by this study, sGC stimulators fail to activate oxidized sGC and thus provide no or limited advantage over PDE-5 inhibitors in restoring erectile function. The finding that BAY 58-2667 was more potent in relaxing corporal tissue expressing apo-sGC provides a solid scientific base for the further development of this new class of drugs because they might have advantages over current therapies for treating ED. This conclusion also is supported by the enhanced erectile responses seen with the administration of a different sGC activator (BAY 60-2270) after sGC oxidation and its ability to restore erectile function in obese mice and in a rat model of cavernosal nerve injury.10,12,38 Moreover, Frey et al39 presented clinical evidence that sGC activators possess erectile properties because they found that spontaneous penile erection was one of the most frequent treatment-emergent adverse events after intake of BAY 58-2667. Similar responses of CC in sGCb1þ/þ and sGCb1ki/ki mice to 8-pCPT-cGMP and forskolin showed that the pathway downstream of sGC was intact in sGCb1ki/ki mice (Figures 3 and 7). These results further illustrate that the abolished sGC-related responses are not due to an aspecific impairment of relaxation related to structural damage or to intrinsic changes in the relaxing properties of sGCb1ki/ki smooth muscle cells. Furthermore, these J Sex Med 2017;14:196e204
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ED in Heme-Deficient NO-Unresponsive Mice
data exclude a compensatory role for the adenylate cyclase and cyclic adenosine monophosphate signaling pathway.40
CONCLUSION This article describes the penile vascular phenotype of a mouse model that allowed us to distinguish between the sGC-dependent and sGC-independent effects of NO. Our data provide evidence that NO exerts its erectile properties solely through sGC activation. The present data also show the therapeutic potential of compounds able to activate apo-sGC in the setting of ED. In addition, our results suggest an important contribution of the lower expressed sGCa2b1 isoform in penile erection. This finding supports the idea of developing agents specifically targeting the sGCa2b1 isoform because this might limit potential side effects and thus provide a therapeutic advantage. The potential of sGC activators to ameliorate erectile function in several disease models and the physiologic pathways involved require further investigation.
ACKNOWLEDGMENTS We thank the animal caretakers for maintaining the animal facility. Corresponding Author: Johan Van de Voorde, PhD, Department of Pharmacology, Ghent University, De Pintelaan 185, Ghent 9000, Belgium. Tel: 32(0)9-332-3342; Fax: 32(0)9-332-8966; E-mail: [email protected] Conflicts of Interest: The authors report no conflicts of interest. Funding: Grants from FWO-Vlaanderen and the Bijzonder Onderzoeksfonds and the Geconcerteerde Onderzoeks Actie of Ghent University.
STATEMENT OF AUTHORSHIP Category 1 (a) Conception and Design Kelly Decaluwé; Johan Van de Voorde (b) Acquisition of Data Kelly Decaluwé (c) Analysis and Interpretation of Data Kelly Decaluwé; Bart Pauwels; Charlotte Boydens; Johan Van de Voorde Category 2 (a) Drafting the Article Kelly Decaluwé; Johan Van de Voorde (b) Revising It for Intellectual Content Kelly Decaluwé; Bart Pauwels; Charlotte Boydens; Robrecht Thoonen; Emmanuel S. Buys; Peter Brouckaert; Johan Van de Voorde
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