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. Author manuscript; available in PMC: 2013 Apr 17.
Published in final edited form as: J Sex Med. 2012 May 23;9(8):1980–1987. doi: 10.1111/j.1743-6109.2012.02798.x

Targeting NADPH Oxidase Decreases Oxidative Stress in the Transgenic Sickle Cell Mouse Penis

Biljana Musicki 1, Tongyun Liu 1, Sena F Sezen 1, Arthur L Burnett 1
PMCID: PMC3435474  NIHMSID: NIHMS381318  PMID: 22620981

Abstract

Introduction

Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown.

Aims

We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis.

Methods

SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67phox, p47phox, and gp91phox), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes.

Main Outcome Measures

Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis.

Results

Relative to hemi mice, SCD increased (P < 0.05) protein expression of NADPH oxidase subunits p67phox, p47phox, and gp91phox, 4-HNE-modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P < 0.05) the abnormalities in protein expressions of p47phox, gp91phox (but not p67phox) and 4-HNE, but only slightly (P > 0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters.

Conclusion

NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in the SCD mouse penis.

Keywords: eNOS Uncoupling, Antioxidants, Priapism

Introduction

Sickle cell disease (SCD) is a hemoglobinopathy resulting from the expression of abnormal sickle hemoglobin (HbS) and consequent red blood cell rigidity, diminished blood flow, and hypoxia. SCD is also characterized by a chronic vasculopathy, involving reduced nitric oxide (NO) bioavailability, enhanced responses to vasoconstrictors, and elevated oxidative stress [1,2].

Oxidative stress, an imbalance between the production and elimination of reactive oxygen species (ROS), has been proposed to be a major factor in the pathogenesis of SCD. ROS, produced in vascular smooth muscle cells and endothelial cells, may induce vascular dysfunction by scavenging NO or affecting eNOS expression and activity, depleting NOS cofactors, generating vasoconstrictors, affecting smooth muscle cell integrity, inactivating antioxidants, and causing structural and functional changes [3]. Increased ROS production in SCD has been attributed to upregulation of NADPH oxidase [4], activation of xanthine oxidase [5], eNOS uncoupling [6,7], as well as autooxidation of HbS [1]. In addition, the protective mechanisms afforded by antioxidants are decreased in SCD. SCD patients and animal models exhibit low levels of vitamins A, C, E, and zinc [8,9] and deficiency of glutathione reductase [10] and glutathione peroxidase (GPx) [11], while superoxide dismutase (SOD) protein expression or activity and catalase expression are either decreased [12,13] or increased [14].

Major clinical manifestations of vasculopathy in SCD include pulmonary hypertension, priapism, and stroke. Priapism is a highly prevalent complication of SCD, affecting about 40% of men with SCD [15–17]. This erection disorder consists of non-willful, excessive, and often recurrent penile erection unrelated to sexual excitement or desire. Ischemic priapism, the most common form of priapism in which blood flow in the corpora cavernosa is absent, is frequently associated with irreversible penile fibrosis and tissue damage, and, if untreated, may lead to permanent and irreversible erectile dysfunction (ED) [18–20].

The mechanisms responsible for priapism are the focus of intense research, but still remain incompletely defined. Priapism has been associated with chronically impaired NO bioavailability in the penis resulting in a defect in the NO/cyclic guanosine monophosphate/phosphodiesterase type 5 signaling pathway [16,21–23], overproduction of adenosine [18,24], and upregulation of opiorphins (enzymes involved in the polyamine synthesis) [25]. The NO depletion mechanism has been attributed to decreased eNOS phosphorylation on Ser-1177 and decreased eNOS interaction with heat-shock protein 90 [26].

Oxidative stress in the SCD penis contributes to endothelial dysfunction and cavernosal tissue damage. Increased lipid peroxidation and protein oxidation in corporal tissue of rats with opiorphin-induced priapism and in transgenic SCD mice have recently been reported [27]. However, the sources of ROS and the mechanisms responsible for ROS formation in the penis remain unknown.

Aims

The aim of this study was to investigate enzymatic sources of ROS, specifically NADPH oxidase and eNOS uncoupling, selected enzymatic antioxidants, and oxidative stress, in the SCD penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress, conceivably improving vascular function in the SCD mouse penis. For this study, we used transgenic SCD mouse model.

Materials and Methods

Animals

Seven to 9 months old male transgenic SCD (sickle) and hemizygous (hemi) mice were used [19]. Transgenic SCD mice with knockout of all mouse hemoglobin genes and expressing exclusively human HbS were developed at Lawrence Berkeley National Laboratory [28]. Sickle and hemi mice are obtained by breeding sickle male mice to hemi females in-house. Sickle and hemi mice were treated with an NADPH oxidase inhibitor apocynin (10 mM, 8.3 mg/day in drinking water, Calbiochem, San Diego, CA, USA) or vehicle for 4 weeks [29]. Flaccid penes were collected before hemi (N = 14) and sickle (N = 14) mice were sacrificed. All experiments were conducted in accordance with the ethical standards of the Johns Hopkins University School of Medicine Guidelines for the Care and Use of Animals.

Western Blot Analysis

Minced penile tissue was homogenized as described [30]. Homogenates were resolved on 4–20% Tris gels and transferred to polyvinylidene difluoride membrane. Membranes were probed with polyclonal rabbit anti-4-hydroxy-2-nonenal (HNE) antibody at 1:2,000 (Alpha Diagnostic International, San Antonio, TX, USA), rabbit anti-p47phox antibody at 1:1,000 (Upstate Biotechnology Inc., Lake Placid, NY, USA), mouse anti-p67phox antibody at 1:600, mouse anti-gp91phox antibody at 1:1,000 (both from BD Transduction Laboratories, San Diego, CA, USA) [29], or rabbit anti-SOD1, SOD2, GPx1, or catalase antibodies (all from Abcam, Cambridge, MA, USA) at 1:1,000 dilutions. Signals were standardized to β-actin. For the analysis of the dimeric and monomeric forms of eNOS, low-temperature sodium dodecyl sulfate (SDS)-gel electrophoresis was used with partially purified penile homogenates, as described previously [31]. Membranes were then probed with rabbit anti-eNOS antibody (BD Transduction Laboratories) at 1:1,000 dilution. Bands were detected by horseradish peroxidase conjugated anti-mouse or anti-rabbit antibodies (GE Health-care, Piscataway, NJ, USA), and analyzed using National Institutes of Health Image software. eNOS uncoupling was represented inversely as a ratio of active eNOS dimers to inactive eNOS monomers. All results were expressed relative to hemi mice treated with vehicle.

Statistical Analysis

Statistical analysis was performed by using one-way analysis of variance, followed by Newman–Keuls multiple comparison test or by t-test when appropriate. The data were expressed as the mean ± standard error of the mean. A value of P < 0.05 was considered to be statistically significant.

Results

Effect of SCD on Protein Expression of Antioxidants GPx1, SOD1, SOD2, and Catalase in the Penis

Protein expression of GPx1, a key antioxidant enzyme, was significantly (P < 0.05) decreased in penes of sickle compared to hemi mice. Protein expression of SOD1, SOD2, and catalase did not differ in penes of sickle compared to hemi mice (Figure 1).

Figure 1.

Figure 1

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Effect of SCD on protein expressions of GPx1, SOD1, SOD2, and catalase in the penis. Left panels are representative Western immunoblots. Right panels represent quantitative analysis of GPx1, SOD1, SOD2, and catalase in penes of hemi and sickle mice. Each bar represents the mean ± SEM of 5–7 mice. *P < 0.05 vs. hemi

Effect of SCD and Apocynin Treatment on Protein Expression of NADPH Oxidase Subunits in the Penis

Protein expressions of NADPH oxidase subunits p67phox, p47phox, and gp91phox were significantly (P < 0.05) increased in penes of sickle compared to hemi mice. Treatment of sickle mice with apocynin, which inhibits the assembly of the subunits within the membrane to an active enzymatic complex and decreases protein expression of NADPH oxidase subunits [29,32], significantly (P < 0.05) decreased protein expressions of p47phox and gp91phox (but not p67phox) in the penis (Figure 2). Apocynin treatment of hemi mice did not affect protein expressions of any of the NADPH oxidase subunits in the penis.

Figure 2.

Figure 2

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Effect of SCD and apocynin treatment on protein expressions of NADPH oxidase subunits in the penis. Upper panels are representative Western immunoblots of the NADPH oxidase subunits in penes of hemi, hemi treated with apocynin, sickle, and sickle mice treated with apocynin. Lower panels represent quantitative analysis of NADPH oxidase subunits p67phox, p47phox, and gp91phox in penes in the same treatment groups. Each bar represents the mean ± SEM of 5–7 mice. *P < 0.05 vs. hemi; **P < 0.05 vs. sickle + vehicle; Vehic = Vehicle; Apoc = Apocynin

Effect of SCD and Apocynin Treatment on eNOS Uncoupling in the Penis

The ratio of eNOS dimer (functional eNOS)/monomer (nonfunctional eNOS), inversely related to eNOS uncoupling, was significantly (P < 0.05) decreased in penes of sickle mice relative to levels found in hemi mouse penes (Figure 3). Apocynin treatment slightly, but not significantly (P > 0.05), increased the dimer/monomer ratio in penes of sickle mice, and had no effect on this ratio in penes of hemi mice.

Figure 3.

Figure 3

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Effect of SCD and apocynin treatment on eNOS uncoupling in the penis. Upper panel is a representative Western immunoblot of the dimers/monomers in penes of hemi, hemi treated with apocynin, sickle, and sickle mice treated with apocynin. Lower panel represents quantitative analysis of eNOS dimer/monomer ratio in penes in the same treatment groups. Each bar represents the mean ± SEM of 6–7 mice. *P < 0.05 vs. hemi; Vehic = Vehicle; Apoc = Apocynin

Effect of SCD and Apocynin Treatment on 4-HNE-Modified Proteins in the Penis

4-HNE is a relatively stable end product of lipid peroxidation used as a biomarker for oxidative damage. It is membrane diffusible and highly reactive, and covalently binds to histidine, lysine, and cysteine residue of proteins, forming adducts that alter protein function and structure [33]. The amount of 4-HNE-modified proteins was significantly (P < 0.05) increased in penes of sickle mice compared with the pattern in hemi mice (Figure 4). Apocynin significantly (P < 0.05) decreased 4-HNE expression in the penis of sickle mice, and had no effect on 4-HNE expression in penes of hemi mice.

Figure 4.

Figure 4

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Effect of SCD and apocynin treatment on 4-HNE-modified proteins in the penis. Upper panel is a representative Western immunoblot of 4-HNE in penes of hemi, hemi treated with apocynin, sickle, and sickle mice treated with apocynin. Lower panel is a quantitative analysis of 4-HNE-modified proteins in penes in the same treatment groups. The analysis of 4-HNE is a densitometric composite of all proteins in each lane. Each bar represents the mean ± SEM of 5–7 mice. *P < 0.05 vs. hemi; **P < 0.05 vs. sickle + vehicle; Vehic = Vehicle; Apoc = Apocynin

Discussion

The present study demonstrates that NADPH oxidase and eNOS uncoupling are sources of ROS in the penis of sickle mice. Deficiency of GPx1 further contributes to oxidative stress by weakening an enzymatic antioxidant potential. Targeting NADPH oxidase decreases oxidative stress in SCD penis, implying a major role for NADPH oxidase as a source of ROS in the SCD penis. Taken together, our data suggest that increased oxidative stress due to upregulation of NADPH oxidase and eNOS uncoupling, and decreased antioxidant GPx1 in the SCD penis, conceivably provides a molecular basis for SCD-associated erectile disorder. The findings further suggest that targeting NADPH oxidase may offer a potential treatment for improving vascular function in the SCD penis, and, conceivably, priapism. This is of special importance, since recent studies point to the greater importance of targeting ROS formation, rather then ROS scavenging, in reducing oxidative damage [34].

Oxidative stress plays a significant role in SCD, contributing to endothelial dysfunction. Activated endothelial cell NADPH oxidase has been implicated in ROS production in the cerebral vasculature of SCD transgenic mice [4]. The NADPH oxidases are a family of enzymes that catalyze electron transfer from cytosolic NADPH or NADH to molecular oxygen to generate superoxide. Of the seven family members, four have been identified as important sources of ROS in the vasculature: Nox1, Nox2, Nox4, and Nox5. The prototype Nox2 (gp91phox)-containing NADPH oxidase possesses cytosolic subunits (p47phox, p67phox, or homologues) and membrane-bound subunits (gp91phox and p22phox), which form a functional enzyme complex upon activation [35]. Increased protein expression of NADPH oxidase subunits has been demonstrated in the penis of diabetic [36,37], hypertensive [38,39], and hypercholesterolemic [29] animals in parallel with increased oxidative stress and ED. We now report that SCD is associated with increased protein expression of NADPH oxidase subunits p47phox, p67phox, and gp91phox in the penis, implying activation of NADPH oxidase in the penis by SCD.

Uncoupled eNOS represents another important source of ROS that may contribute to SCD vasculopathy. Under physiologic conditions, NOS isoforms structurally reside as homodimeric oxidoreductases. This form permits their oxidative catalysis of L-arginine to NO. In the uncoupled state, electron transfer within the active site of eNOS becomes “uncoupled” from L-arginine oxidation, and results in oxidation of molecular oxygen and preferential superoxide production over NO production [40]. Moreover, superoxide is rapidly converted into highly toxic nitrogen species such as peroxynitrite, if NO is also present locally. Peroxynitrite causes cellular dysfunction through oxidative damage to DNA, proteins and lipids, nitration of tyrosines, release of vasoconstrictors, and further eNOS uncoupling [41]. A major cause of eNOS uncoupling involves deficiency of the NOS cofactor tetrahydrobiopterin (BH4) due to its decreased synthesis and/or increased oxidation [42]. eNOS-derived superoxide has been implicated in exaggerated blood cell–endothelial cell interaction in the cerebral microcirculation of SCD mice [6] through mechanisms possibly involving BH4 deficiency. Elevated inactive eNOS monomers have also been reported in the lungs of transgenic SCD mice, and may be associated with pulmonary hypertension [7]. Loss of the active dimeric form of eNOS and predominance of inactive monomeric eNOS may explain compensatory upregulation of eNOS protein expression, yet decreased NO bioavailability, described in a transgenic SCD mouse model [43]. Uncoupled eNOS has recently been demonstrated to be a source of ROS in the penis of aged [44] and hypercholesterolemic [29,31] animals. We now report decreased ratio of functional eNOS dimers/nonfunctional eNOS monomers in the penis of SCD transgenic mice vs. their hemi counterparts in parallel with increased oxidative stress, implying uncoupled eNOS as a source of ROS in the SCD penis. It is important to note that eNOS uncoupling plays a key role in both the loss of NO biosynthetic capacity, and the increase in endothelial oxidative stress, both contributing to vascular dysfunction in the SCD penis.

In isolated endothelial cells [45], aorta of hypertensive mice [46,47], and penis of hypercholesterolemic mice [29], ROS produced by NADPH oxidase initiate eNOS uncoupling. In order to determine whether NADPH oxidase is an upstream activator of eNOS uncoupling in the penis of sickle mice, we treated these mice with apocynin. Apocynin is a specific inhibitor of NADPH oxidase activation, which both prevents assembly of cytosolic units with the membrane complex and negatively regulates NADPH oxidase subunits expression [29,32]. We present data showing that inhibition of NADPH oxidase by apocynin decreased protein expressions of p47phox and gp91phox subunits, as expected, and decreased lipid peroxidation in the penis of SCD mice. Apocynin treatment also slightly reduced eNOS uncoupling, implying that ROS generated by NADPH oxidase are partially, although not solely, responsible for eNOS uncoupling. These findings invoke a novel role for NADPH oxidase as a major source of ROS in the SCD penis. The lack of reversal of eNOS coupling by NADPH oxidase inhibition indicates that other ROS sources may also contribute to eNOS uncoupling in the SCD penis. The role for xanthine oxidase in ROS production has been demonstrated in the lungs of SCD mice [5]. Furthermore, other mechanisms for eNOS uncoupling, which are unrelated to oxidation of the enzyme or its cofactor BH4 [48], may also operate in the SCD penis. They may include decreased BH4 synthesis or decreased availability of the NOS substrate L-arginine. Suboptimal endothelial arginine levels have been reported in the lungs of SCD mice, reflecting increased arginase activity [7] and elevated asymmetric dimethylarginine (ADMA) [49]. Future studies are needed to explore the exact mechanism of eNOS uncoupling in the SCD penis.

The pro-oxidant environment in the SCD penis may be due not only to high production of ROS, but also to a reduction in antioxidant systems. SOD, which converts superoxide to hydrogen peroxide, exists in three isoforms: cytoplasmic copper-zinc (SOD1), mitochondrial manganese (SOD2), and extracellular copper-zinc (SOD3). Catalase, mainly expressed in peroxisomes, catalyzes the decomposition of hydrogen peroxide to water and molecular oxygen. GPx reduces hydrogen peroxide to water, and lipid peroxides to their corresponding alcohols [50]. Eight different isoforms of GPx have been discovered. GPx1 is the ubiquitous isoform expressed in the cytosol and mitochondria in most cells, including the endothelium. Recent studies indicated a critical role of GPx1 in preserving NO bioavailability and protecting the cardiovascular system against oxidative stress [51,52]. The few studies that have examined antioxidant enzymes in patients or mice with SCD have yielded contradictory results with respect to levels of SOD and catalase [11,13]. Our studies demonstrate unaltered protein expressions of SOD1, SOD2, and catalase, and decreased protein expression of GPx1 in the SCD mouse penis. Low levels of GPx1 may promote susceptibility to oxidative stress by allowing accumulation of harmful oxidants, thus further contributing to eNOS uncoupling, increased oxidative stress, and conceivably endothelial dysfunction.

A limitation of this study is that we only investigated the molecular changes in ROS sources, selective enzymatic antioxidants, and oxidative stress, in the SCD mouse penis. In vivo erectile studies will be performed in the future to evaluate whether the priapic phenotype is decreased by NADPH oxidase inhibition, which would corroborate our findings.

Conclusions

SCD increases protein expressions of NADPH oxidase subunits, induces eNOS uncoupling, and decreases antioxidant GPx1 in the SCD penis, resulting in increased oxidative stress. Apocynin decreases oxidative stress through inhibition of NADPH oxidase upregulation, implying a major role for NADPH oxidase as a ROS source in the SCD penis. The findings suggest that targeting a specific ROS-generating source in the penis may offer a potential treatment for SCD-related priapism.

Acknowledgments

This work was supported by NIH/NIDDK grant RO1DK067223 to ALB.

Footnotes

Conflict of Interest: None.

Statement of Authorship

  • Category 1
    1. Conception and Design
      Biljana Musicki; Arthur L. Burnett
    2. Acquisition of Data
      Biljana Musicki; Tongyun Liu; Sena F. Sezen
    3. Analysis and Interpretation of Data
      Biljana Musicki; Arthur L. Burnett
  • Category 2
    1. Drafting the Article
      Biljana Musicki
    2. Revising It for Intellectual Content
      Biljana Musicki; Tongyun Liu; Sena F. Sezen; Arthur L. Burnett
  • Category 3
    1. (Final Approval of the Completed Article
      Biljana Musicki; Tongyun Liu; Sena F. Sezen; Arthur L. Burnett

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