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The Journal of Pharmacology and Experimental Therapeutics logoLink to The Journal of Pharmacology and Experimental Therapeutics
. 2016 Nov;359(2):230–237. doi: 10.1124/jpet.116.235473

Beneficial Effect of the Nitric Oxide Donor Compound 3-(1,3-Dioxoisoindolin-2-yl)Benzyl Nitrate on Dysregulated Phosphodiesterase 5, NADPH Oxidase, and Nitrosative Stress in the Sickle Cell Mouse Penis: Implication for Priapism Treatment

Fábio H Silva 1,✉, Serkan Karakus 1, Biljana Musicki 1, Hotaka Matsui 1, Trinity J Bivalacqua 1, Jean L dos Santos 1, Fernando F Costa 1, Arthur L Burnett 1
PMCID: PMC5074485  PMID: 27540002

Abstract

Patients with sickle cell disease (SCD) display priapism, and dysregulated nitric oxide (NO) pathway may contribute to this condition. However, current therapies offered for the prevention of priapism in SCD are few. The 3-(1,3-dioxoisoindolin-2-yl)benzyl nitrate (compound 4C) was synthesized through molecular hybridization of hydroxyurea and thalidomide, which displays an NO-donor property. This study aimed to evaluate the effects of compound 4C on functional and molecular alterations of erectile function in murine models that display low NO bioavailability, SCD transgenic mice, and endothelial NO synthase and neuronal NO synthase double gene-deficient (dNOS−/) mice, focusing on the dysregulated NO–cGMP– phosphodiesterase type 5 (PDE5) pathway and oxidative stress in erectile tissue. Wild-type, SCD, and dNOS−/− mice were treated with compound 4C (100 μmol/kg/d, 3 weeks). Intracavernosal pressure in anesthetized mice was evaluated. Corpus cavernosum tissue was dissected free and mounted in organ baths. SCD and dNOS−/− mice displayed a priapism phenotype, which was reversed by compound 4C treatment. Increased corpus cavernosum relaxant responses to acetylcholine and electrical-field stimulation were reduced by 4C in SCD mice. Likewise, increased sodium nitroprusside–induced relaxant responses were reduced by 4C in cavernosal tissue from SCD and dNOS−/− mice. Compound 4C reversed PDE5 protein expression and reduced protein expressions of reactive oxygen species markers, NADPH oxidase subunit gp91phox, and 3-nitrotyrosine in penises from SCD and dNOS−/− mice. In conclusion, 3-week therapy with the NO donor 4C reversed the priapism in murine models that display lower NO bioavailability. NO donor compounds may constitute an additional strategy to prevent priapism in SCD.

Introduction

Sickle cell disease (SCD) is a blood disorder, multisystem disease, that afflicts millions worldwide, caused by a single-nucleotide substitution (GTG for GAG) in the sixth codon of the β-globin gene (Rees et al., 2010). Men with SCD display a severe alteration of erectile function, priapism, which is defined as a penile erection that persists beyond, or is unrelated to, sexual interest or stimulation (Salonia et al., 2014). This condition constitutes a urological emergency requiring prompt diagnosis and treatment because it is associated with erectile tissue damage and erectile dysfunction (Salonia et al., 2014). In male patients with SCD, the prevalence of priapism is up to 45% and the rate of resultant erectile dysfunction exceeds 30% (Adeyoju et al., 2002; Olujohungbe et al., 2011; Lionnet et al., 2012). Despite its high prevalence rate, current therapies offered for this condition are nonpreventive and often administered too late during an episode of priapism. Currently, few therapies focus on the prevention of priapic events in patients with SCD. Studies that focus on the development of new preventive therapies are of great clinical relevance. Alterations of the nitric oxide (NO)–cGMP–phosphodiesterase-5 (PDE5) signaling pathway have been implicated as a major mechanism for priapism in SCD, which is not simply a consequence of vascular blockage (Kato, 2012; Anele et al., 2015).

NO is well established as a mediator of penile erection. NO, released from nitrergic nerves and endothelial cells, activates the soluble guanylate cyclase enzyme in cavernosal smooth muscle, resulting in the accumulation of intracellular cGMP, which leads to relaxation of corpus cavernosum smooth muscle and penile erection (Burnett, 2006). Intracellular cGMP is rapidly inactivated to 5′GMP by PDE5, thus ceasing the erectile response (Carson and Lue, 2005). In Berkeley transgenic SCD mice, lower basal NO production leads to decreased PDE5 protein expression in the penis (Champion et al., 2005). SCD mice exhibit an amplified corpus carvenosum relaxation response in vitro (Mi et al., 2008; Claudino et al., 2009), as well as prolonged penile erection and priapism (Champion et al., 2005) mediated by the NO–cGMP signaling pathway. In penises from SCD patients with priapism, decreased PDE5 protein expression was also observed (Lagoda et al., 2013). Thus, when an erectile stimulus occurs in vivo, cGMP accumulates in cavernosal smooth muscle cells, rendering the penile vasculature uncontrollably dilated, and penile erection persists (i.e., priapism) as cGMP is not degraded as a consequence of PDE5 dysregulation. Endothelial nitric oxide synthase (eNOS) gene-deficient mice and combined eNOS and neuronal NO synthase (nNOS) double gene-deficient (dNOS−/−) mice also display a priapism phenotype associated with downregulation of PDE5 protein expression (Champion et al., 2005; Lagoda et al., 2014) and exhibit exacerbated cavernosal relaxations induced by the NO donor sodium nitroprusside (Nangle et al., 2004).

Oxidative stress has been reported to play an important role in the pathophysiology of priapism in SCD (Musicki et al., 2012; Bivalacqua et al., 2013). NADPH oxidase is a multisubunit flavoprotein complex consisting of the cytosolic subunits p40phox, p47phox, and p67phox; small GTPase Rac; and membrane-bound subunits gp91phox and p22phox (Frey et al., 2009). The subunit gp91phox acts as a crucial catalytic subunit for superoxide anion production (Frey et al., 2009). Increased NADPH oxidase subunit gp91phox expression was observed in penises from SCD mice (Musicki et al., 2012) and SCD patients with priapism (Lagoda et al., 2013). Excess superoxide anion reacts rapidly with NO, decreasing NO bioavailability and forming peroxynitrite, which is a powerful oxidant and cytotoxic agent (Pacher et al., 2007). Recently, upregulation of nitrotyrosine, a product of protein tyrosine nitration mediated by reactive nitrogen species, was shown to be associated with priapism in SCD mice (Bivalacqua et al., 2013; Lagoda et al., 2014).

Due to the lack of available drugs to prevent SCD symptoms, 3-(1,3-dioxoisoindolin-2-yl)benzyl nitrate (compound 4C) was synthesized through molecular hybridization of hydroxyurea and thalidomide (dos Santos et al., 2011). Specifically, NO resulting from the bioconversion of hydroxyurea was inserted into nitrate ester subunits to produce novel hybrid derivatives possessing a NO-donor property, as well as analgesic and anti-inflammatory properties (dos Santos et al., 2011). In this study, because low NO bioavailability leads to priapism, we hypothesized that long-term administration of compound 4C may reverse the erectile function alterations that are associated with dysregulated NO–cGMP–PDE5 signaling and increased oxidative stress in the penises from transgenic SCD and dNOS−/− mice. Therefore, in the present study, we have undertaken functional and molecular studies to evaluate the beneficial effects of 3-week i.p. administration of compound 4C on priapism and oxidative stress markers in the penis from SCD and dNOS−/− mice.

Materials and Methods

Animals and Treatment.

Three- to 5-month-old wild-type (WT), SCD transgenic, and dNOS−/− male mice were used. Transgenic SCD mice express exclusively human sickle hemoglobin and were generated by knockout of mouse α and β globins and insertion of a single transgene that expresses human α and β S globin (Pászty et al., 1997). SCD mice are obtained by interbreeding sickle cell males with hemizygous females in-house. Breeding pairs for SCD mice (strain number 3342) and WT mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Genotyping was performed by Transnetyx (Cordova, TN). Adult male dNOS−/− mice were originally developed on a BL6/129 hybrid background strain and backcrossed 12 generations on a C57BL/6 strain. Age-matched WT C57BL/6 males were used as controls because this represents the predominant background strain for the transgenic mice. SCD, dNOS−/−, and WT male mice were treated with compound 4C (100 μM/kg/d) or vehicle (20% Cremophor) daily for 3 weeks via i.p. injection. All animal procedures were conducted in accordance with the ethical standards of the Johns Hopkins University School of Medicine Guidelines for the Care and Use of Laboratory Animals.

Intracavernosal Pressure Measurement.

Mice were anesthetized with isoflurane (1–1.5%) inhalation. The right carotid artery was cannulated with PE-10 tubing and attached to a pressure transducer to measure mean arterial pressure (MAP) (Lagoda et al., 2014). Surgical procedures required freeing the shaft of the penis of skin and fascia and removal of part of the ischiocavernous muscle to expose the crus. A 30-gauge needle filled with 250 U/ml heparin and connected to polyethylene tubing (PE-10) was inserted into the right crura and connected to a pressure transducer to allow continuous measurement of intracavernosal pressure (ICP). A midline abdominal incision exposed the prostate, where the right major pelvic ganglion and cavernous nerve were identified posterolateral to the prostate. For electrically stimulated penile erections, a bipolar electrode attached to an S48 stimulator (Grass Instruments, Quincy, MA) was placed around the cavernous nerve, and stimulation parameters were 4 V at a frequency of 16 Hz with square-wave duration of 5 ms. ICP was recorded using the DI-190 system (Dataq Instruments, Akron, OH) from the beginning of the experiment when the cannula was inserted into the crura continuously until the animal was euthanized (Lagoda et al., 2014). Erectile function was represented by the maximal ICP response during electrical stimulation (ICP at the plateau), ICP response after stimulation [poststimulated area under the curve (AUC)], as well as half detumescence time, as recorded using the MATLAB program (Mathworks, Natick, MA), and parameters were normalized per MAP.

In Vitro Functional Assays and Concentration–Response Curves in Mouse Isolated Corpus Cavernosum.

Strips of corpus cavernosum obtained from mice anesthetized with ketamine and xylazine were mounted in a 5-mL organ system containing Krebs-Henseleit solution (mM: 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11 glucose) at 37°C and continuously bubbled with a mixture of 95% O2 and 5% CO2 (pH 7.4). Changes in isometric force were recorded using a strip myograph for isometric force recording (model 610M; Danish Myo Technology, Aarhus, Denmark) coupled with an acquisition system (PowerLab 8/30, LabChart 7; ADInstruments, Sydney-NSW, Australia). The resting tension was adjusted to 2.5 mN at the beginning of the experiments. The equilibration period was 60 minutes, and the bathing medium was changed every 15 minutes until the start of the experiments. Cumulative concentration–response curves were constructed for both the muscarinic agonist acetylcholine (ACh; 10−9 to 3 × 10−6 M) and the NO-donor compound sodium nitroprusside (SNP; 10−8 to 10−4 M) in tissue strips precontracted with phenylephrine (10−5 M).

Electrical-Field Stimulation in Corpus Cavernosum Strips.

Electrical-field stimulation (EFS) was applied to the cavernosal strips placed between two platinum electrodes connected to a Grass S88 stimulator (Astro-Med Industrial Park, Warwicks, RI). EFS was conducted at 50 V, 1-ms pulse width, and trains of stimuli lasting 10 seconds at varying frequencies. Frequency–response relationships were investigated at supra maximum voltage in all preparations stimulated electrically. To study the nitrergic cavernosal relaxations, tissues were pretreated with guanethidine (3 × 10−5 M; to deplete the catecholamine stores of adrenergic fibers) and atropine (10−6 M; to produce muscarinic antagonism) prior to precontraction with phenylephrine. When a stable contraction level was attained, a series of EFS-induced relaxations was constructed (1–32 Hz) (Silva et al., 2014).

Data Analysis of Functional Assays.

Nonlinear regression analysis to determine the pEC50 was carried out using GraphPad Prism (GraphPad Software, San Diego, CA) with the constraint that F = 0. All concentration–response data were fitted to a logistic function in the following form: E = Emax/[(1 + [10c/10x]n) + F], where E is the maximum response produced by agonists; Emax is the maximal response; c is the logarithm of the EC50, the concentration of drug that produces a half-maximal response; x is the logarithm of the concentration of the drug; the exponential term, n, is a curve fitting parameter that defines the slope of the concentration–response line, and F is the response observed in the absence of added drug. Relaxing responses were calculated as percentages of the maximal changes from the steady-state contraction produced by phenylephrine. Data are shown as the percentage of relaxation of n experiments, expressed as the mean values ± S.E.M.

Western Blot Analysis.

Penile tissue was homogenized as described (Hurt et al., 2002). Total penile homogenates (50 μg total protein) were run on 4–20% Tris-HCl gels (Bio-Rad Laboratories, Hercules, CA), transferred to a polyvinylidene fluoride membrane, and incubated overnight at 4°C with the following antibodies: polyclonal anti-PDE5 (1:500; Abcam, Cambridge, MA), polyclonal anti–vasodilator-stimulated phosphoprotein (VASP; 1:1000; Cell Signaling Technology, Danvers, MA), polyclonal anti-VASP phosphorylated at Ser239 (1:1000; Cell Signaling Technology), monoclonal anti–3-nitrotyrosine (1:1000; Abcam), monoclonal anti-gp91phox (1:1000; BD Transduction Laboratories, San Diego, CA), and monoclonal anti–β-actin (1:7000; Sigma-Aldrich, St. Louis, MO) (Lagoda et al., 2014). Quantified densitometry results were set as a ratio to β-actin and normalized to the control group (WT) probed on the same gel, making the control group 1 arbitrary unit. Quantified densitometry results of VASP phosphorylated at Ser239 were normalized to total VASP.

Drugs and Chemicals.

Compound 4C was synthesized by the Laboratory of Research and Development of New Drugs (Lapdesf; State University of São Paulo, Araraquara, SP, Brazil) (dos Santos et al., 2011). The purity determined by analytical methods was found to be superior to 98.5%. ACh, atropine, guanethidine, phenylephrine, and sodium nitroprusside were obtained from Sigma-Aldrich. All reagents used were of analytical grade. Stock solutions were prepared in deionized water. Dilutions were prepared immediately before use.

Statistical Analysis.

The program GraphPad Prism (GraphPad Software) was used for statistical analysis. Data are expressed as the mean ± S.E.M. of N experiments. Statistical comparisons were made using one-way analysis of variance, and the Tukey method was chosen as a post-test. Student’s unpaired t test was also used when appropriate. A value of P < 0.05 was considered statistically significant.

Results

Effect of Long-Term Treatment with Compound 4C on Priapism in SCD Mice.

Cavernous nerve stimulation (4 V) caused increases of ICP/MAP ratio in all groups (Fig. 1A). Maximal ICP/MAP ratio values were similar among WT and SCD mice (Fig. 1A). No significant changes after compound 4C treatment were observed in maximal ICP/MAP ratio values of WT-4C and SCD-4C groups (Fig. 1A). However, half detumescence time was significantly (P < 0.05) higher in SCD compared with the WT group, indicating that SCD mice display exaggerated erectile responses (Fig. 1B). Three-week treatment with compound 4C normalized (P < 0.05) half detumescence time, with no significant changes in the WT group (Fig. 1B). Poststimulated ICP/MAP ratio values after 9 minutes were significantly (P < 0.05) higher in SCD compared with the WT group, which were also reversed by long-term treatment with compound 4C (Fig. 1C). No significant changes after compound 4C treatment were observed in poststimulated ICP/MAP ratio values of the WT group (Fig. 1C). Typical traces of ICP in SCD-vehicle and SCD-4C are shown in Fig. 1D.

Fig. 1.

Fig. 1.

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Cavernous nerve (CN) electrical stimulation (4 V) induced changes in (A) max ICP over MAP, (B) half-normalized detumescence time, and (C) max ICP over MAP in WT and SCD mice treated or not with compound 4C (100 μmol/kg/d, 3 weeks) 9 minutes poststimulation. (D) Representative ICP tracing in response to CN electrical stimulation in SCD-V and SCD-4C. Data represent the mean ± S.E.M. for five to eight mice in each group. *P < 0.05 versus WT-V. #P < 0.05 SCD-V versus SCD-4C. Stim, stimulation; V, vehicle.

Effect of Long-Term Treatment with Compound 4C on Priapism in dNOS−/− Mice.

Cavernous nerve stimulation (4 V) caused increases of ICP/MAP ratio in all groups (Fig. 2). However, maximal ICP/MAP ratio values were higher in dNOS−/− compared with WT mice, which were reversed by long-term treatment with compound 4C (Fig. 2A). No significant changes after compound 4C treatment were observed in maximal ICP/MAP ratio values of WT. dNOS−/− mice display uncontrolled erectile responses, indicated by increased poststimulated AUC/MAP ratio values (Fig. 2B). Compound 4C treatment significantly reduced (P < 0.05) poststimulated AUC/MAP ratio values in the dNOS−/−-4C group. AUC/MAP ratio values were not determined in WT-vehicle and WT-4C groups. Typical traces of ICP in dNOS−/−-vehicle and dNOS−/−-4C mice are shown in Fig. 2, C and D, respectively. Compound 4C treatment did not change MAP in WT, SCD, and dNOS−/−. MAP (mmHg) values of WT-vehicle, WT-4C, SCD-vehicle, SCD-4C, dNOS−/−-vehicle, and dNOS−/−-4C mice were 105 ± 5, 102 ± 5, 88 ± 7, 74 ± 15, 115 ± 7, and 120 ± 4, respectively.

Fig. 2.

Fig. 2.

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Cavernous nerve (CN) electrical stimulation (4 V) induced changes in (A) max ICP over MAP and (B) total ICP over MAP in WT and SCD mice treated or not with compound 4C (100 μmol/kg/d, 3 weeks) 9 minutes poststimulation. (C and D) Representative ICP tracing in response to CN electrical stimulation in dNOS−/−-V and dNOS−/−-4C. Data represent the mean ± S.E.M. for five to eight mice in each group. *P < 0.05 versus WT-V. #P < 0.05 SCD-V versus SCD-4C. Stim, stimulation; V, vehicle.

Concentration–Response Curves to ACh, SNP, and EFS in Cavernosal Tissue: Effect of Prolonged Treatment with Compound 4C.

The cumulative addition of ACh (10−9 to 3 × 10−6 M) to phenyleprine-contracted tissues produced concentration-dependent relaxations in all groups. ACh potency (pEC50) value was significantly higher (P < 0.05) in the corpus cavernosum of SCD compared with WT mice (Fig. 3, A and D), which was reversed by long-term treatment with 4C (Fig. 3, C and D). No significant changes after compound 4C treatment were observed in ACh-induced corpus cavernosum relaxations of WT mice (Fig. 3B). pEC50 values for ACh are shown in Fig. 3D.

Fig. 3.

Fig. 3.

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Concentration–response curves to ACh (A–C) and relaxation responses to EFS (E) in corpus cavernosum strips from WT and SCD mice, treated or not with compound 4C (100 μmol/kg/d, 3 weeks). (D) Potency (pEC50) values for ACh in all groups. Data were calculated relative to the maximal changes from the contraction produced by phenylephrine (10−5 M) in each tissue, which was taken as 100%. Data represent the mean ± S.E.M. for five mice in each group. *P < 0.05 versus WT-V. #P < 0.05 versus SCD-V. V, vehicle.

EFS of cavernosal tissues pretreated with guanethidine (3 × 10−5 M) and atropine (10−6 M) caused frequency-dependent corpus cavernosum relaxations in WT and SCD groups (Fig. 3E). Corpus cavernosum relaxations to EFS were significantly higher (P < 0.05) in SCD compared with WT mice, as observed at 2 and 8 Hz. Long-term treatment with compound 4C reduced (P < 0.05) the EFS-induced increased relaxant responses in corpus cavernosum from SCD, particularly at 2, 4, and 8 Hz (Fig. 3E). No significant changes after 4C treatment were observed in EFS-induced corpus cavernosum relaxations of WT mice (Fig. 3E).

The cumulative addition of SNP (10−8 to 10−4 M) also produced concentration-dependent relaxations in all groups (Fig. 4). The maximal response (Emax) induced by SNP was significantly higher (P < 0.05) in corpus cavernosum from SCD (93 ± 6%) and dNOS−/− (105 ± 1.5%) compared with WT mice (72 ± 5%; Fig. 4A), which was fully restored to WT values with compound 4C treatment (Fig. 4, B and C). No significant changes after compound 4C treatment were observed in SNP-induced corpus cavernosum relaxations of WT mice (Fig. 4D). No significant differences of pEC50 for SNP were found among WT-vehicle (6.13 ± 0.06), WT-4C (6.02 ± 0.07), dNOS−/−-vehicle (6.07 ± 0.04), dNOS−/−-4C (6.00 ± 0.04), and SCD-vehicle groups (6.02 ± 0.08). However, the SNP pEC50 value was significantly lower (P < 0.05) in SCD treated with compound 4C (5.71 ± 0.08) compared with SCD-vehicle.

Fig. 4.

Fig. 4.

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Concentration–response curves to SNP in corpus cavernosum strips from WT (A and D), SCD (A and B), and dNOS−/− mice (A and C), treated or not with compound 4C (100 μM/kg/d, 3 weeks). Data were calculated relative to the maximal changes from the contraction produced by phenylephrine (10−5 M) in each tissue, which was taken as 100%. Data represent the mean ± S.E.M. for 5–11 mice in each group. *P < 0.05 versus WT-V. #P < 0.05 versus respective vehicle group. V, vehicle.

Effect of Long-Term Compound 4C Treatment on Protein Expression of PDE5 in Penis from SCD and dNOS−/− Mice.

The protein expression of PDE5 was significantly reduced (P < 0.05) by approximately 48% and 35% in penile tissues from SCD and dNOS−/−, in comparison with the WT group, respectively (Fig. 5). Treatment with compound 4C significantly increased (P < 0.05) the protein level of PDE5 in penises from SCD and dNOS−/− groups, with no significant changes in the WT-4C group (Fig. 5).

Fig. 5.

Fig. 5.

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Representative images of Western blotting (top panels) and protein values (bottom panels) for PDE5 in homogenates of penises from WT and SCD (A) and WT and dNOS−/− (B) mice, treated chronically or not with compound 4C (100 μmol/Kg, 3 weeks). Data represent the mean ± S.E.M. for 4–11 mice in each group. A single band for PDE5 was normalized to β-actin. *P < 0.05 versus WT-V. #P < 0.05 versus respective vehicle group. V, vehicle.

Effect of Long-Term Compound 4C Treatment on Protein Expression of VASP Phosphorylated at Ser239 in the Penis from SCD Mice.

VASP phosphorylation at Ser239 was significantly reduced (P < 0.05) by 37% in penile tissues from the SCD group in comparison with the WT group (Fig. 6). Treatment with compound 4C significantly increased (P < 0.05) VASP phosphorylation at Ser239 in the penises from SCD group, with no significant changes in WT-4C group (Fig. 6).

Fig. 6.

Fig. 6.

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Representative images of Western blotting (top panels) and protein values (bottom panels) for VASP phosphorylated at Ser239 (p-VASP) in homogenates of penises from WT and SCD mice, treated chronically or not with compound 4C (100 μmol/Kg, 3 weeks). Data represent the mean ± S.E.M. for five mice in each group. A single band for p-VASP was normalized to VASP. *P < 0.05 versus WT-V. #P < 0.05 versus respective vehicle group. V, vehicle.

Effect of Long-Term Compound 4C Treatment on Protein Expressions of gp91phox and 3-Nitrotyrosine in the Penis from SCD and dNOS−/− Mice.

The protein expression of gp91phox was significantly higher (P < 0.05) by approximately 44% and 79% in cavernosal tissues from SCD and dNOS−/− groups, in comparison with the WT group, respectively (Fig. 7, A and B). Treatment with compound 4C significantly reduced (P < 0.05) the protein level of gp91phox in the SCD group (Fig. 7A). Although there was a tendency toward decreased protein expressions for gp91phox in the dNOS−/− group treated with compound 4C, it did not achieve statistical significance (Fig. 7B). The protein expression for 3-nitrotyrosine was significantly higher (P < 0.05) by approximately 80% and 60% in cavernosal tissues from SCD and dNOS−/−, in comparison with the WT group, respectively (Fig. 7, C and D). Compound 4C treatment significantly reduced (P < 0.05) the protein level of 3-nitrotyrosine by approximately 64% and 25% in SCD and dNOS−/− groups (Fig. 7, C and D). In WT mice, the protein expressions for gp91phox and 3-nitrotyrosine were not affected by compound 4C treatment.

Fig. 7.

Fig. 7.

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Representative images of Western blotting (top panels) and protein values (bottom panels) for gp91phox (A and B) and 3-nitrotyrosine (3-NT; C and D) in homogenates of penises from WT, SCD, and dNOS−/− mice, treated chronically or not with compound 4C (100 μmol/Kg, 3 weeks). The analysis of 3-NT is a densitometric composite of all proteins in each lane. Data represent the mean ± S.E.M. for 5–11 mice in each group. Bands for gp91phox and 3-NT were normalized to β-actin. *P < 0.05 versus WT-V. #P < 0.05 versus respective vehicle group.

Discussion

In SCD, one of the most common inherited blood disorders worldwide with approximately 300,000 affected new births per year, children and adults display priapism, a known complication (Piel et al., 2013). Low NO bioavailability in the penis leads to priapism in SCD mice, eNOS gene-deficient mice, or combined eNOS and nNOS gene-deficient (dNOS−/−) mice (Champion et al., 2005). Therefore, considering that compound 4C possesses a NO donor property (dos Santos et al., 2011), we decided to evaluate the effects of compound 4C treatment on priapism, in two murine models that display low NO bioavailability, SCD and dNOS−/− mice (Champion et al., 2005; Claudino et al., 2009). In our study, we initially evaluated the ICP in anesthetized mice as a direct index of penile erection. Stimulation of the cavernous nerve caused an exaggerated erectile response in SCD and dNOS−/− mice, which was reversed by compound 4C treatment. In the penis, nNOS is primarily responsible for initiation of normal erection, whereas both nNOS and eNOS help maintain normal sustained penile erection (Hurt et al., 2012). Therefore, we next evaluated endothelium-dependent and nitrergic relaxations in corpus cavernosum. In accordance with our in vivo findings, enhanced relaxant responses to ACh and EFS in SCD mouse corpus cavernosum were reversed by compound 4C treatment. To our knowledge, our study is the first to show that long-term treatment with a NO donor compound prevents functional alterations of the penis from SCD mice.

The NO–cGMP signaling pathway is the most important pathway for mediating erectile function (Burnett, 2006). The concentration of cGMP in corpus cavernosal smooth muscle cells is dependent on the balance between its production by soluble guanylate cyclase and its breakdown by PDE5 (Carson and Lue, 2005). Selective PDE5 inhibition elevates intracellular cGMP levels and enhances NO-dependent cavernosal relaxations (Burnett, 2008). Consistent with our functional findings, the expression of PDE5 was reduced in cavernosal tissue from SCD and dNOS−/− mice, which was restored by treatment with compound 4C. cGMP is a positive regulator of PDE5 gene expression in cavernosal smooth muscle (Lin et al., 2002). In our study, upregulation of PDE5 by compound 4C treatment may reflect enhanced cGMP production in the erectile tissue from SCD and dNOS−/− mice. Protein kinase G is activated by cGMP and preferentially phosphorylates VASP at Ser239 (Francis et al., 2010). Phosphorylation of VASP at Ser239 is considered a reliable biomarker for monitoring the NO-stimulated cGMP–protein kinase G pathway (Oelze et al., 2000). In fact, in our study, phosphorylation of VASP at Ser239 was increased by compound 4C treatment in penises from SCD mice, indicating increased cGMP levels. Our results are consistent with a molecular study that reported that a NO donor compound increases PDE5 and VASP phosphorylation at Ser239 in the penis from SCD mice (Lagoda et al., 2014). A limitation of our study is that we did not measure cGMP in penises of SCD and dNOS−/− mice, due to a limited amount of tissue. A previous study reported that transfection of eNOS gene-deficient mice with an adenovirus encoding eNOS resulted in normalization of PDE5A protein and activity as well as correction of priapic activity (Champion et al., 2005). In the WT group, 3-week treatment with compound 4C affected neither erectile responses nor protein expression of PDE5 nor VASP phosphorylation at Ser239.

NO can be exogenously supplied to tissues and cells by various NO-generating compounds. The inorganic compound SNP is an agent that releases NO in biologic systems by nonenzymatic and enzymatic mechanisms (Kowaluk et al., 1992; Bonaventura et al., 2008). In our study, SNP-induced cavernosal relaxations were significantly increased in SCD and dNOS−/− groups, which were restored to WT values by treatment with compound 4C. This is consistent with our findings showing that protein expression of PDE5 is lower in the penis from SCD and dNOS−/− groups, which was increased by treatment with compound 4C. Thus, it is likely that amplifications of SNP-induced relaxations reflect excess accumulation of cGMP in corpus cavernosum smooth muscle due to lower degradation of cGMP by PDE5 in SCD and dNOS−/− mice, thus favoring exaggerated erectile responses. In penises from SCD mice, upregulation of opiorphins and increased production of the vasodilator adenosine are also associated with the pathophysiology of priapism (Mi et al., 2008; Kanika et al., 2009). A recent study showed that excess adenosine can lead to downregulation of PDE5 in SCD mouse penises (Ning et al., 2014).

SCD is associated with increased oxidative stress, an imbalance between the production and elimination of reactive oxygen species (Nur et al., 2011). The NADPH oxidase complex is a major source of superoxide anion in vascular cells. NADPH oxidase is a multienzyme oxidant complex composed of several subunits, in which gp91phox acts as a crucial catalytic subunit for superoxide anion production (Frey et al., 2009). In SCD mice, NADPH oxidase has been associated with cerebral microvascular dysfunction (Wood et al., 2005) and appears to play an important role in priapism (Musicki et al., 2012). In our study, elevated gp91phox and 3-nitrotyrosine protein expressions in penile tissues from SCD and dNOS−/− were detected, which is consistent with previous studies (Musicki et al., 2012; Bivalacqua et al., 2013). In mouse erectile tissue, the soluble guanylyl cyclase stimulator BAY 41-2272 reduces superoxide anion formation through inhibition of NADPH oxidase activity and reduction of NADPH oxidase subunit gp91phox expression, an effect mediated by a cGMP-dependent mechanism (Teixeira et al., 2007; Nunes et al., 2015). Likewise, the PDE5 inhibitor sildenafil inhibits p47phox NADPH oxidase subunit expression and superoxide anion formation in cavernosal smooth muscle cells (Koupparis et al., 2005). In contrast, the NO donor DETA-NONOate inhibits NADPH oxidase-dependent superoxide anion production by a cGMP-independent mechanism in human endothelial cells, without changing the protein expression of gp91phox (Selemidis et al., 2007). In our study, compound 4C treatment reduced the protein expressions of gp91phox and 3-nitrotyrosine in the penis from SCD and dNOS−/− mice. Although there was a trend toward decreased protein expression of gp91phox in the dNOS−/− group treated with compound 4C, it did not achieve statistical significance. Therefore, in this context, it is reasonable to suggest that the reduction of gp91phox seen after a 3-week treatment with compound 4C in SCD and dNOS−/− mice may be through a cGMP-dependent mechanism. Because superoxide anion can react with NO, generating peroxynitrite, it is likely that decreased 3-nitrotyrosine protein expression by compound 4C may be associated with lower superoxide anion production by NADPH oxidase in smooth muscle cavernosal cells of SCD and dNOS−/− mice. Unfortunately, we have not evaluated the effects of treatment with compound 4C on levels of reactive oxygen species in penises from SCD and dNOS−/− mice. In penises from SCD mice, uncoupled eNOS and xanthine oxidase also appear to be an important source of reactive oxygen species (Bivalacqua et al., 2013). A previous study reported that 3-week therapy with sildenafil prevents priapism in SCD mice via control of oxidative and nitrosative stress in penises, as well as through normalization of PDE5 activity (Bivalacqua et al., 2013).

In summary, our results show that 3-week therapy with the NO donor compound 4C ameliorated the low NO bioavailability-associated priapism due to upregulation of protein expression of PDE5 and downregulation of NADPH oxidase subunit gp91phox and nitrosative stress in the penis from SCD and dNOS−/− mice. Therefore, NO donor compounds may constitute an additional strategy to prevent priapism in SCD because SCD-related priapism therapies are few.

Abbreviations

ACh

acetylcholine

AUC

area under the curve

dNOS−/−

eNOS and nNOS double gene-deficient

EFS

electrical-field stimulation

eNOS

endothelial NO synthase

ICP

intracavernosal pressure

MAP

mean arterial pressure

nNOS

neuronal NO synthase

NO

nitric oxide

PDE5

phosphodiesterase type 5

SCD

sickle cell disease

SNP

sodium nitroprusside

VASP

vasodilator-stimulated phosphoprotein

WT

wild-type

Authorship Contributions

Participated in research design: Silva, Costa, Burnett.

Conducted experiments: Silva, Karakus.

Contributed new reagents or analytic tools: Matsui, Bivalacqua, dos Santos, Burnett.

Performed data analysis: Silva, Karakus, Musicki, Costa, Burnett.

Wrote or contributed to the writing of the manuscript: Silva, Costa, Burnett.

Footnotes

This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01DK067223 to A.L.B.] and São Paulo Research Foundation (Fundaçao de Amparo à Pesquisa do Estado de Sao Paulo) [Grants 2010/12495-6, 2013/19781-2, 2014/21965-7, and 2014/00984-3 to F.H.S. and F.F.C.].

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