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- Review -
NADPH oxidase: recent evidence for its role in erectile dysfunction
Liming Jin, Arthur L. Burnett
Department of Urology, The James Buchanan Brady Urological Institute, Johns Hopkins Medical Institutions,Baltimore,
MD 21287, USA
Abstract
Important roles for reactive oxygen species (ROS) in physiology and pathophysiology have been increasingly
recognized. Under normal conditions, ROS serve as signaling molecules in the regulation of cellular functions.
However, enhanced ROS production as a result of the activation of nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase contributes significantly to the pathogeneses of vascular diseases. Although it has become evident
that increased ROS is associated with erectile dysfunction (ED), the sources of ROS in the penis remain largely
unknown. In recent years, emergent evidence suggests the possible role of NADPH oxidase in inducing ED. In this
review, we examine the relationship between ROS and ED in different disease models and discuss the current
evidence basis for NADPH oxidase-derived ROS in ED. (Asian J Androl 2008 Jan; 10: 6_13)
Keywords: reactive oxygen species; erectile function; superoxide; penis; nitric oxide
Correspondence to: Dr. Liming Jin, Department of Internal Medicine, University of California, Davis, CA 95616, USA.
Tel: +1-530-752-2140 Fax: +1-530-752-3470
E-mail: lmjin@ucdavis.edu
DOI: 10.1111/j.1745-7262.2008.00371.x
1 Introduction
Erectile dysfunction (ED) affects millions of men worldwide and reduces quality of life [1]. Although ED may
result from psychological, neurological, and hormonal defects, vascular impairment accounts for a major portion of
male ED [2, 3]. ED is often associated with chronic vascular diseases such as atherosclerosis, hypertension, and
heart disease. Elucidation of erectile mechanisms has led to the discoveries of therapeutic targets, among which nitric
oxide (NO) has been recognized as a critical molecule in erection physiology. Sexual stimuli induce the release of NO
from penile nerve endings and endothelial cells, which in turn relaxes corpus cavernosal smooth muscle and increases
blood flow to penis. Studies have shown that increased reactive oxygen species (ROS) reduce NO production or
bioavailability, leading to impaired endothelial function and erectile function [4_7].
ROS include free radicals such as superoxide and hydroxyl radicals and non-radicals such as hydrogen peroxide.
Abundant evidence has demonstrated the importance of ROS in physiologic functions and the pathogeneses of various
diseases. ROS are produced either through non-enzymatic ways or through enzymatic systems such as nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, un-coupled endothelial NO synthase, cytochrome
P450, and the mitochondria respiratory chain. Under normal conditions, cells are capable of self-protecting against
the continuous formation of low levels of ROS via major intracellular antioxidant enzymes including superoxide
dismutase (SOD), catalase, glutathione peroxidase, and glutathione reductase. For example, studies have shown that
cavernous endothelial cells are able to detoxify ROS within a well-defined range using these enzymes
[8]. These antioxidant enzymes are usually up-regulated as a compensatory mechanism when ROS production is increased [5,
9]. However, sustained high levels of ROS generation will diminish antioxidant enzyme activities and increase
oxidative stress, leading to cell damage.
The effects of ROS on erectile function have been investigated with most of the emphasis placed on the activities
of antioxidant enzymes. However, the sources of ROS remain largely unknown. It has become evident that NADPH
oxidase is a major source for ROS formation in the vascular wall. In recent years, emergent evidence also suggests
the possible role of NADPH oxidase in inducing ED. In the first part of this review, we will briefly examine the
relationship between ROS and ED in different disease
models. In the second part, we will focus on the basic
molecular actions of NADPH oxidase and discuss the
current knowledge of NADPH oxidase-derived ROS in ED.
2 ROS and ED
2.1 Hypertension
Most forms of hypertension are characterized by
impaired endothelium-dependent vasodilation, which is
partly due to an increase in ROS production. Results
from clinical studies indicate the high prevalence of ED
in hypertensive patients when compared to the general
population [2, 10]. In experimental hypertensive animal
models, ED has been demonstrated in both spontaneously
hypertensive rats (SHR) and deoxycorticosterone
acetate-salt hypertensive rats [11, 12]. The levels of
thiobarbituric acid reactive substances (TBARS), an indicator for
oxidative stress, were increased in SHR penes, leading
to reduced NO-dependent relaxations of isolated cavernosal smooth muscle [6]. Diminished SOD
activity was observed in SHR rat penes when compared to
that of Wistar-Kyoto rats, which may be one of the causes
for this increase in ROS production in SHR.
2.2 Diabetes
Diabetes mellitus is the most common risk factor for
ED [13]. The risk of ED is three-fold greater for
diabetic men and occurs at an earlier age than non-diabetic
men. The link between ROS and diabetes-associated
ED has been investigated. One possible source of ROS
is from circulating monocytes. Morano et
al. [14] reported that ROS generated by monocytes were
significantly increased in patients with ED when compared to
those of patients without ED. In streptozotocin
(STZ)-induced type I diabetic rats, endothelial NO synthase and
neuronal NO synthase-mediated relaxations of cavernosal
muscle strips are reduced [4]. Treatment with the
antioxidant, α-lipoic acid, partially restores the relaxations,
suggesting increased ROS production in diabetic penes.
Another group studied the effects of ROS during the
progression of diabetes. They found that increased ROS
formation, indicated by increased TBARS levels and
reduced glutathione levels, were associated with impaired
ED in STZ-induced diabetic rats. More interestingly,
oxidative stress is further increased in rats with
long-term diabetes which is correlated with more severe ED
when compared to rats with short-term diabetes [15].
Protein kinase C (PKC) is an important molecule that
regulates a variety of cellular functions such as smooth
muscle contraction [16]. Hyperglycemia increases ROS
production and protein expression of PKC isoforms in
cultured rat cavernosal smooth muscle cells [17]. The
up-regulation of PKC is dependent on ROS because
exposure of these cells with the antioxidant, vitamin E,
prevents this increase. Taken together, these data suggest
that ROS contribute significantly to diabetes-associated
ED. This conclusion is further supported by the
evidence that gene transfer of extracellular-SOD reduces
superoxide production and restores erectile function in
STZ-induced diabetic rats [18]. Data are lacking at
present to suggest that ROS production contributes to
ED in type 2 diabetes.
2.3 Hypercholesterolemia and hyperlipidemia
High levels of ROS were detected in cavernosal strips
from hypercholesterolemic rabbits which is correlated
with impaired endothelium-dependent relaxation but not
endothelium-independent relaxation [5]. Increased ROS
production may increase oxidized low density lipoprotein,
leading to enhanced contractility of cavernosal strips [19].
Although it is not clear whether hypercholesterolemic
rabbits have impaired erectile function in
vivo, these data provide some evidence to support the clinical
observations that hypercholesterolemia, hyperlipidemia and
subsequently developed atherosclerosis are risk factors for
ED [20].
2.4 Hyperhomocysteinemia
A high plasma concentration of homocysteine is an
independent risk factor for the development of
cardiovascular diseases such as atherosclerosis.
Homocysteine-induced endothelial injury is associated with elevated
ROS production [21_23]. The adverse effects of homocysteine were demonstrated in isolated rabbit
cavernosal strips. Pre-incubation of cavernosal strips isolated
from normal rabbits with homocysteine decreases endothelium-dependent relaxation responses, which are
reversed by SOD or catalase [24]. Similarly, cavernosal
strips isolated from hyperhomocysteinemic rabbits also
display impaired endothelium-dependent relaxation along
with increased ROS formation [25]. Copper exaggerated the ability of homocysteine to produce ROS through
the Fenton reaction. Hyperhomocysteinemic rabbits
having received penicillamine, a copper-chelator, have
reduced superoxide generation in penes and better
endothelium-dependent relaxation [25].
2.5 Aging
The prevalence of ED is significantly increased from
5% in men aged between 20 to 39 years to more than
70% in men aged 70 years and above [26]. Strong
evidence has supported the concept that ROS are the key
players of the aging process. Increased ROS levels were
detected in aged Brown-Norway rat penes by
lucigenin-enhanced chemiluminescence and dihydroethidium (DHE)
[27]. Moreover, increased oxidative stress is implicated
with a decreased ratio of the reduced form of glutathione
versus oxidized glutathione in aged penes [28]. It is
associated with decreased SOD protein expression in
penes from aged rats when compared to that of young
rats [28]. In vivo adenoviral gene transfer of
extracellular-SOD restores erectile responses to cavernous nerve
and agonist- stimulation to the levels observed in young
rats [27]. These data suggest that ROS play significant
roles in the development of aging-associated ED.
2.6 Nerve injury
Nerve injury-related ED is common after radical
prostatectomy. Using a unilateral cavernous nerve
injury rat model, our group demonstrated that nerve injury
induces oxidative stress in rat penile tissue indicated by
increased nitrotyrosine staining [9]. The injury induces
a rapid compensatory response in penile tissue by
increasing glutathione peroxidase protein expression.
However, this effect is not sustained in saline-treated nerve
injured rats. Treatment with FK506, a nerve protecting
agent, maintains the high levels of glutathione peroxidase
expression and improves erectile function [9]. Consistently,
studies from another group showed that increased
oxidative stress and ED occurred within three weeks in the
rats that had unilateral nerve dissection [29]. Resection
of the cavernous nerve causes more severe injury, which leads
to worse erectile function and greater ROS formation when
compared to that of rats underwent nerve dissection
[29].
3 NADPH oxidase
3.1 NADPH oxidase subunits
NADPH oxidase, a multicomponent enzyme, catalyzes the production of superoxide using an electron
derived from NADPH. Superoxide is converted to
hydrogen peroxide by SOD which subsequently leads to the
formation of other ROS such as hydroxyl radicals.
NADPH oxidase was first identified in phagocytes.
It consists of the catalytic subunit
gp91phox (phox, phagocyte oxidase) and regulatory subunits
p22phox, p47phox,
p67phox, and p40phox (Figure
1) [30, 31]. gp91phox contains
a flavin adenine dinucleotide (FAD) and two heme groups
and provides binding sites for NADPH and oxygen
molecule. It exists together with
p22phox as a stabilized membrane-bound complex called cytochrome
b558 [32]. p47phox,
p67phox, and p40phox are located in the cytosol in
the resting state. In addition, a small GTPase protein,
Rac, participates in the assembly of the NADPH oxidase
complex and is required for NADPH oxidase activation,
Once stimulated, the cytosolic subunits migrate to
the membrane, where they assemble with
gp91phox and p22phox [33]. The active oxidase generates superoxide by
transferring the electron from NADPH to oxygen.
3.2 Activation of NADPH oxidase
NADPH oxidase is activated by endothelin-1,
angiotensin II (Ang II), thrombin, platelet-derived growth
factor, tumor necrosis factor-α (TNF-α) and cytokines
through receptor-mediated mechanisms [34_37]. Mechanical stress and phorbol-12-myristate-13-acetate
(PMA) can also directly activate NADPH oxidase
[38_40]. Phosphorylation of p47phox on several serine
residues is a crucial step to initiate the translocation of
cytosolic components to the membrane and activate NADPH
oxidase [41], although some data suggest that only
Ser379 is essential for oxidase activity and membrane
association [42]. The phosphorylation causes a
conformational change of p47phox and enables the two SH3
(Src homology 3) domains of
p47phox to bind to the proline-rich region on the carboxyl terminus of
p22phox [43]. In addition, p47phox
has a Phox-homology (PX) domain which binds to phosphoinositides on the membrane,
contributing to membrane anchoring of
p47phox. p47phox facilitates
the recruitment of p67phox to the membrane through tail
to tail interactions between its proline-rich region and the
SH3 domain of p67phox. Therefore,
p47phox functions as an organizer.
p67phox is considered an activator because
its main function is to transfer the electron from NADPH
to FAD [44, 45]. This is a rate-limiting step in
superoxide generation. The function of
p40phox is less known and controversial. Some studies suggest that
p40phox is not required for high level superoxide production or even
down-regulates NADPH oxidase activity [46, 47]. In
contrast, other reports indicate that
p40phox enhances superoxide production through improving the efficiency of
p67phox in the activation of NADPH oxidase [48, 49].
Activation of NADPH oxidase also requires a small
GTP-binding protein-Rac. Rac is a member of the Rho
family, and three isoforms of Rac have been identified:
Rac1, Rac2 and Rac3. Rac1 and Rac3 are universally
expressed except that Rac3 is not expressed in neutrophils.
Rac2 is only expressed in hematopoietic cells. Rac
functions as a molecular switch cycling between the
GDP-bound inactive form and the GTP-bound active form.
This process is tightly regulated by three proteins:
GDP-dissociation inhibitor (GDI), Rho guanine exchange
factor (RhoGEF), and GTPase activating protein (GAP).
GDP-bound Rac is stabilized by GDI in the cytosol of
the resting cells. During activation, RhoGEF facilitates
the exchange of GTP for GDP, leading to the migration
of Rac to the membrane along with the core cytosolic
NADPH oxidase subunits. GAP inactivates Rac by increasing the intrinsic GTP hydrolytic activity of Rac.
Studies have shown that p67phox has a tetratricopeptide
repeat domain serving as a docking site specific for
Rac [50].
3.3 Homologues of NADPH oxidase
Recently, gp91phox and its novel homologues were
found in various types of cells other than phagocytes.
These homologues are termed NOX (NADPH oxidase). NOX1,
gp91phox (or NOX2), NOX3, NOX4 and NOX5
are expressed in smooth muscle cells, endothelial cells,
fibroblasts and cancer cells [51, 52]. NOX1 through
NOX4 have very similar structures. However, studies
have shown that NOX4 activation requires only the
presence of p22phox and not any cytosolic subunits [53].
Because NOX5 has an extra unique domain containing four
binding sites for calcium, the regulation of NOX5 is
dependent on intracellular calcium concentration and does
not require any other subunits [54, 55].
In addition, homologues of p47phox, NOX organizer 1
(NOXO1) and p67phox, NOX activator 1 (NOXA1) were
discovered in colon epithelial cells [56, 57].
Co-expression of NOXO1 and NOXA1 greatly increase the ability
of NOX1 to generate superoxide.
3.4 Localization of NADPH oxidase
3.4.1 Endothelial cells
NOX2 and its regulators p22phox,
p47phox, p67phox and Rac1 were all found in endothelial cells isolated from
animals as well as human coronary artery and umbilical
vein [58, 59]. In addition, NOX1, NOX3, and NOX4 were demonstrated in mouse lung endothelial cells [60,
61]. Recently, NOX5 variants NOX5β, NOX5δ and NOX5S were identified in human microvascular
endothelial cells [62].
3.4.2 Smooth muscle cells
In addition to NOX2, p22phox,
p47phox, p67phox, Rac1, and novel NOX family members were demonstrated in
human and animal smooth muscle cells [63]. mRNA and protein expressions of NOX1 and NOX4 were
detected in mouse lung smooth muscle cells, rat aortic
smooth muscle cells and human aortic smooth muscle
cells [64_66]. It is not known whether NOX3 and NOX5
are expressed in smooth muscle cells.
3.4.3 Fibroblast
NOX2, p22phox, p47phox, and
p67phox were detected in rat and rabbit aortic adventitial fibroblasts [67, 68]. NOX4
and NOX5 were predominantly expressed in human cardiac fibroblasts while NOX1 and NOX2 were barely
detectable [69].
4 Physiologic role of NADPH oxidase
Phagocytes generate high levels of ROS to kill
bacteria as a self-defense mechanism through activation of
NADPH oxidase. In non-phagocytic cells, a small amount
of NADPH oxidase-derived ROS is generated at physiologic levels, functioning as second messengers in
response to cellular stimuli. ROS are involved in the
regulation of cell growth and proliferation, cell migration,
angiogenesis and apoptosis through activation of various
signaling pathways. The actions of ROS on smooth muscle cell growth and proliferation in response to growth
factors and hormones are mainly mediated by
mitogen-activated protein kinases (MAPK) such as ERK1/2,
p38MAPK, c-Jun N-terminal kinase [70, 71]. Protein
kinase B (Akt) is another downstream molecular target
of ROS. Hingtgen et al. [72] reported that ROS
generated by NADPH oxidase activates Akt, which play a key
role in Ang II-induced cardiomyocyte hypertrophy.
NADPH oxidase also participates in cytoskeletal
reorganization through interaction with the orphan adaptor
TRAF4 and focal contact scaffold Hic-5 in lamellipodia
at the leading edge of the cells, which is essential for cell
migration [73, 74]. During the process of angiogenesis,
NADPH-derived ROS not only up-regulate vascular endothelial growth factor (VEGF) expression, but also
mediate VEGF induced phosphatidylinositol 3-kinase
(PI3K)/ERK1/2 signaling cascade via VEGF receptor-2
[61, 75, 76]. In addition, NADPH oxidase participates
in the control of cell cycle arrest and apoptosis via
activation of cell cycle inhibitors
p21cip1 and p53 [77].
5 Pathophysiologic role of NADPH oxidase in ED
Lower levels of ROS are important in the regulation
of physiologic function. However, excessive
production of ROS may overwhelm the cellular antioxidant
defense mechanisms, leading to pathologic changes
observed in vascular diseases. ROS cause vascular
damages by promoting smooth muscle cell growth and
proliferation, increasing extracellular matrix protein
deposition, activating matrix metalloproteinases,
inducing endothelial dysfunction, and altering vascular tone.
It has been well established that enhanced NADPH
oxidase activity contributes to the development of
atherosclerosis, hypertension, diabetes and hypercholesterolemia. The
role of NADPH oxidase in ED is vastly understudied
compared to other vascular diseases.
Although increased ROS production is linked to ED
in different disease models, the sources of ROS have
never been investigated in vivo. Some initial
in vitro experiments provide indirect evidence for the possible
involvement of NADPH oxidase in the development of
ED. Greater superoxide production is detected in penile
tissue isolated from hypercholesterolemic rabbits than
that in control rabbit penes [78]. NADPH oxidase
inhibitors apocynin and diphenyleneiodonium chloride (DPI)
significantly reduce not only the basal production of
superoxide in control rat penes but also that in
hypercholesterolemic penes. Inhibitors of xanthine oxidase, NO
synthase and the mitochondrial electron transport chain
have no effect on superoxide formation. Apocynin and
DPI also partially restore the impaired
endothelium-dependent smooth muscle relaxation. These data suggest
that NADPH oxidase-derived ROS are produced at low
levels in normal penes and enhanced NADPH oxidase activity is responsible for ED in hypercholesterolemic
rabbits. The phosphodiesterase 5 (PDE-5) inhibitor NCX
911 recovers cavernosal relaxation partly through
inhibition of NADPH oxidase activity.
Another study demonstrated that the NADPH oxidase subunit
p47phox is expressed in rabbit cavernosal
smooth muscle cells [79]. Incubation with U46619, a
thromboxane mimetic, induces ROS production along with increased
p47phox protein expression. The PDE-5 inhibitor sildenafil reduces ROS in smooth cells by
reducing NADPH oxidase subunit p47phox expression.
Teixeira et al. [80] recently examined the role of NADPH
oxidase in mouse penes. NADPH oxidase-dependent superoxide generation is significantly increased after
incubating mouse cavernosal tissue with U46619 for 1 h
or 8 h. This effect is reversed by the soluble guanylyl
cyclase activator, BAY 41-2272, through decreasing
protein expression of p22phox and
gp91phox. On the other hand, NADPH oxidase-derived ROS may regulate
PDE-5 protein expression. TNF-α and nicotine stimulate ROS
production and increase PDE5 protein expression in
rabbit cavernosal smooth cells [81]. Apocynin, SOD,
catalase as well as sildenafil reduce PDE-5 expression through
inhibition of ROS formation [81]. These data suggest
that NO signaling inhibits NADPH oxidase activity.
Conversely, ROS generated from NADPH oxidase negatively regulates the NO pathway.
Although these in vitro studies suggest that NADPH
oxidase activity is increased in disease states or can be
stimulated by agonists, it remains unclear whether and
to what extent NADPH oxidase functions in
vivo in the penis or whether it plays an important role in the
pathogenesis of ED. We recently performed a series of
experiments designed to investigate the role of NADPH
oxidase in the development of ED in hypertension [82].
Consistent with other studies showing that hypertensive
rats exhibit ED [11, 12], in our Ang II-infused hypertensive
rats (4 weeks infusion of subpressor doses of Ang
II), erectile function was significantly impaired. ROS generation
was significantly increased in penes isolated from Ang
II-infused hypertensive rats accompanied by increased
NADPH oxidase subunit p47phox protein expression.
Chronic treatment with apocynin reduced NADPH oxidase protein expression and ROS levels in Ang II-infused
hypertensive rat penes. Correspondingly, preserved
erectile function in hypertensive rats by apocynin treatment
provides further evidence that elevated NADPH oxidase
activity is an important mechanism for
hypertension-associated ED. Furthermore, we investigated the
downstream targets of ROS. We found that increased ROS
formation in Ang II-infused hypertensive rat penes led to
up-regulation of RhoA protein expression, a key
component in the RhoA/Rho-kinase pathway of contractile
mechanism in the penis. ROS also decreased endothelial
NO synthase expression in hypertensive rat penes.
Apocynin treatment reversed these changes to the levels
similar to that of control penes (unpublished data, Jin
et al.). This is the first study to our knowledge that
demonstrated a relationship between NADPH oxidase-derived
ROS and ED at an in vivo level.
6 Conclusion
Accumulating evidence has demonstrated the
importance of NADPH oxidase-derived ROS in both
physiologic and pathophysiologic processes. There is a
dynamic balance between antioxidant systems and ROS
generating systems within cells. Cells may be protected
against the unfavorable effects caused by moderate
increases in ROS production through compensatory mechanisms of up-regulated antioxidant enzyme activity.
However, excessive production of ROS in response to
pathogenic stimuli destroys these antioxidant mechanisms
and causes cell damage. In recent years, the link
between elevated ROS levels and ED has been established
and therapeutic strategies have centered on restoring the
antioxidant ability of cells in the penis. However,
another important aspect is blockage of sources of ROS
formation since ROS forms are extremely active and
interchangeable. It may not be completely effective to
simply scavenge one of the ROS. Therefore, it is crucial
to investigate the mechanisms of ROS generation. Ours
and other studies have demonstrated that NADPH oxidase represents a major source for ROS in the
development of ED. Yet, further characterization of NADPH
oxidase and molecular targets of ROS in penile tissue are
essential to obtain a better understanding of the
pathogenesis of ED and develop pharmacological and
molecular interventions.
Acknowledgement
We acknowledge the support from the National Institute of Health (DK073531 to Liming Jin and DK67223
to Arthur L. Burnett) and American Heart Association
(0530007N to Liming Jin).
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