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- Original Article -
Homocysteine and copper interact to promote type 5
phosphodiesterase expression in rabbit cavernosal smooth
muscle cells
Matthew Hotston1, Jamie Y.
Jeremy2, Jonathon Bloor2, Nick S.
Greaves2, Raj Persad1, Gianni
Angelini2, Nilima Shukla2
1Department of Urology,
2Department of Cardiac Surgery, University of Bristol, Bristol BS8 1TH, UK
Abstract
Aim: To study the effects of homocysteine and copper on type 5 phosphodiesterase (PDE5) expression in cavernosal
vascular smooth muscle cells (CVSMCs) and to investigate superoxide
(O2·-) derived from nicotinamide adenine
dinucleotide phosphate oxidase as homocysteine and copper generate
O2·-, and
O2·- upregulates PDE5 expression.
Methods: CVSMCs derived from rabbit penis were incubated with homocysteine or copper chloride with or without
superoxide dismutase (SOD), catalase, sildenafil citrate, or apocynin (nicotinamide adenine dinucleotide phosphate
inhibitor) for 16 h. The expression of PDE5 and of glyceraldehyde-3-phosphate dehydrogenase (internal standard)
was assessed using Western blot analysis. In parallel,
O2·- was measured spectrophotometrically.
Results: CuCl2 alone (up to
10 μmol/L) and homocysteine alone (up to
100 μmol/L) had no effect on
O2·- formation in CVSMCs
compared to controls. In combination, however, homocysteine and
CuCl2 markedly increased
O2·- formation, an effect blocked by SOD, catalase, apocynin, and sildenafil
(1 μmol/L) when co-incubated over the same time course.
PDE5 expression was also significantly increased in CVSMCs incubated with homocysteine and
CuCl2, compared to controls. This effect was also negated by 16-h co-incubation with SOD, catalase, apocynin and
sildenafil. Conclusion: This represents a novel pathogenic mechanism underlying ED, and indicates that the therapeutic actions of prolonged
sildenafil use are mediated in part through inhibition of this pathway.
(Asian J Androl 2008 Nov; 10: 905_913)
Keywords: erectile dysfunction; superoxide; PDE5; sildenafil
Correspondence to: Mr Matthew Hotston, Department of Urology, University of Bristol, 13 Freeland Place, Bristol BS8 4NP, UK.
Tel: +44-01173-739-049 Fax: +44-01924-881-228
E-mail: matthotston@hotmail.com
Received 2007-07-03 Accepted 2007-11-25
DOI: 10.1111/j.1745-7262.2008.00380.x
1 Introduction
The nitric oxide (NO)_cyclic guanosine
monophosphate (cGMP) system, by eliciting relaxation of
cavernosal smooth muscle and pudendal arteries, is central to
penile erection [1]. NO is released from non-adrenergic
non-cholinergic fibers that innervate the penis and
pudendal arteries and from the endothelium within the
corpus cavernosum and pudendal vasculature [1]. NO then
activates guanylyl cyclase, which catalyses the
conversion of guanosine triphosphate to cGMP, which then
induces vascular smooth muscle relaxation, principally
through reducing calcium mobilisation [1]. Impairment
of NO bioavailability is associated with erectile
dysfunction (ED) [2, 3].
Risk factors for ED associated with reduced NO
include diabetes mellitus (DM), hypertension, dyslipidemia,
smoking, and more recently with hyperhomocysteinemIa
(HHC), all of which are also associated with increased
formation of superoxide
(O2·-) [4, 5].
O2·- reacts with
NO leading to reduced "NO drive" [4, 5]. A principal
intravascular source of
O2·- is nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase [5]. Many
factors associated with ED increase the expression of
NADPH oxidase. These include thromboxane
A2, cytokines, angiotensin II, hypoxia, and
O2·- itself [5, 6]. NADPH oxidase is also linked to the risk factors for ED
listed above [5]. Furthermore, sildenafil inhibits the
activity and expression of NADPH oxidase (and therefore
O2·-) in vascular smooth muscle cells through a
cGMP_protein kinase G (PKG)-mediated mechanism [7_11],
exemplifying the possible central role of NADPH oxidase
in the etiology of ED.
The bioeffectiveness of the NO_cGMP system is also
determined by the inactivation of cGMP by type 5
phosphodiesterases (PDE5) [12_15]. PDE5 hydrolyses cGMP
to inactive guanosine monophosphate (GMP) [16], thereby stopping the dilator effects of NO. The
importance of PDE5 in erection is exemplified by the
therapeutic benefit of sildenafil
(Viagra?; Pfizer, NY, USA), a PDE5 inhibitor, in treating ED [12_15]. A recent study
showed that nicotine and tumor necrosis factor-α
increase the expression of PDE5 through a mechanism
triggered by O2·- derived from NADPH oxidase [17] and
suggested that risk factors for ED might entail an
upregulation of PDE5 by oxidative stress.
Experimental HHC, a risk factor for cardiovascular
disease, also promotes ED in rabbit through
augmentation of O2·- formation and negation of NO bioactivity, an
effect mediated by an upregulation of NADPH oxidase
[18]. Ex vivo, homocysteine alone at concentrations far
above those seen in HHC has little effect on cavernosal
relaxation, indicating that homocysteine alone is not an
independent risk factor for ED [19]. At concentrations
seen in HHC, however, homocysteine interacts with physiological concentrations of copper to inhibit
NO-mediated relaxation of cavernosal and arterial tissue [19,
20]. Penicillamine, a copper chelator, reverses ED
elicited by HHC [9]. It has therefore been suggested that the
erectopathic impact of HHC is mediated by an
interaction of homocysteine with copper that augments
O2·- formation and reduces NO bioavailability. As
O2·- upregulates the expression of PDE5, it is reasonable to suggest that
homocysteine and copper might interact to promote ED
through increased intracavernosal PDE5 expression. In
order to test this possibility, we studied the interactive
effect of homocysteine and copper on
O2·- formation, determined its source, and studied its effects on PDE5
expression. The effect of sildenafil on these systems
was also studied, as sildenafil inhibits the formation of
O2·- through suppression of NADPH oxidase activity
[7_11] and, as such, would be expected to block these
hypothetical sequelae.
2 Materials and methods
New Zealand White rabbits were used for this study,
and given humane care in compliance with Bristol
University and the UK Home Office rules and regulations.
They were killed with a lethal dose of barbiturates, and
the penile tissue promptly excised. The corpora
cavernosae were dissected from the surrounding tunica
albuginea, cut into 2 mm2 segments, and placed in
Dulbecco's modified Eagle's medium (DMEM) with 10%
fetal calf serum (both from Gibco BRL, Paisley, UK)
and incubated at 37ºC in a 95% air, 5%
CO2 incubator for 7 days, during which time cavernosal vascular smooth
muscle cells (CVSMCs) grew from the explant. The explants were removed and adherant CVSMCs trypsinized
and placed in culture plates until confluent. These cells
stained positively for alpha-actin but not von Willebrand
Factor (vWF) or lectin (markers of endothelial cells).
CVSMCs were subsequently incubated at 37ºC in a 95%
air, 5% CO2 incubator as previously described [6]. When
confluent, CVSMCs were quiesced in low glucose DMEM (containing 100 units/mL penicillin and
100 μg/mL streptomycin; Gibco BRL). After 72-h quiescence, the
following experiments were undertaken.
2.1 Effects of homocysteine and copper on
O2·- formation
CVSMCs were incubated with homocysteine
(1 μmol/L_1 mmol/L; Sigma Chemical, Poole, Dorset,
UK), or copper chloride (0.1 μmol/L_10 μmol/L; Sigma Chemical)
or homocysteine plus copper chloride in 4.5 g/L DMEM
for 16 h. O2·- formation was measured using the
reduction of ferricytochrome c method [21, 22].
Supernatants were discarded, and cells washed with sterile
Dulbecco's phosphate-buffered saline (Bio Whittacker,
Bio Science, Erembodegen, Belgium). The cells were
equilibrated with DMEM without phenol red (Bio Whittaker, Bio Science) with or without copper_zinc
superoxide dismutase (SOD; 500 U/mL; Sigma Chemical).
Following 30 min incubation at 37ºC in a 95% air, 5%
CO2 incubator (Heraeus Equipment, Essex, UK),
20 μmol/L horse heart cytochrome c (Sigma Chemical)
was added, and incubated for a further 1 h. The amount of
cytochrome c reduced by SOD was determined at 550 nm
using an Anthos Lucy 1 spectrometer (Laboratory-tech
International, East Sussex, UK) and converted to
μmol/L of O2·- (using the equation
ΔE = 550
nm = 21.1 mM-1 cm
-1 as the extinction coefficient). This reflects the actual
O2·- release [9]. Following determination of the optimal
concentration at which homocysteine plus copper induced
O2·- production, the experiment was repeated with
co-incubation for 16 h with SOD (500 U/mL; converts
O2·- to
H2O2; Sigma Chemical)) and catalase (100 U/mL;
converts H2O2 to
H2O; Sigma Chemical). In order to ascertain the source of increased
O2·- release from NADPH
oxidase, the experiment was repeated with the NADPH
oxidase inhibitor, apocynin (1 μmol/L; Sigma Chemical).
The effects of sildenafil citrate (1 μmol/L for
16 h; Schering Pharmaceuticals, Newbury, UK) on
O2·- release were assessed through
O2·- experiments as previously
described [21, 22].
2.2 Effects of homocysteine and copper on PDE5
expression using Western blot analysis
CVSMCs were incubated with
homocysteine plus copper chloride with or without SOD (500 U/mL),
catalase (100 U/mL), and sildenafil citrate
(1 μmol/L) in 4.5 g/L DMEM for 16 h at 37ºC in a 95% air, 5%
CO2 incubator. For Western blots, the supernatant was
discarded, and the cells were washed with Dulbecco's
phosphate-buffered solution (Gibco BRL). Following the
addition of protein lysis buffer (Tris buffer,
100 mmol/L, pH 6.8) containing 1% glycerol and 1% sodium dodecyl
sulphate, the cell lysates were stored at _20ºC. Protein
content on the lysates was quantified using the MicroBCA
kit (Pierce, Rockford, IL, USA). Samples of equal
protein concentration were loaded to 10% Tris-glycine
sodium dodecyl sulphate gel, as well as rainbow markers
(14_220 kDa; Amersham International, Little Chalfont,
UK) to assess molecular weight, and electophoresed.
Samples were transferred to a Hybond-C
nitro-cellulose-pure membrane (Amersham International). After
blocking in 5% Marvel (Spalding, UK) the membranes were
incubated with 1:1 000 rabbit anti-PDE5 antibody (Cell
Signalling Technology, Hitchin, UK) in 5% bovine serum
albumin (Sigma Chemical) and 0.1% sodium azide at
5ºC, with constant agitation, for 16 h. Following incubation
with the secondary antibody, goat anti-mouse antibody
conjugated with horseradish peroxidase (1:5 000; Dako,
Ely, UK) for 1 h, the blots were developed using
enhanced chemiluminescence (Amersham International) on
X-ray film, and bands scanned using Quantity
One® 1-D analysis software (Bio-Rad Laboratories, Hercules, CA,
USA).
2.3 Data analysis and expression
Data were expressed as mean ± SEM of six separate
studies. The paired t-test was used to compare effects
of homocysteine and CuCl2 with the control. Multiple
group comparisons were made using the repeated measures ANOVA test. Data was calculated using Instat
Graphpad software (Graphpad Software, San Diego, CA,
USA). Significance was accepted at P < 0.05.
3 Results
Homocysteine alone and copper chloride alone
elicited no effects on O2·-
formation by CVSMCs following a 16-h incubation compared to controls (Figure 1). In contrast,
when combined together, homocysteine and copper
chloride markedly augmented the formation of
O2·- (Figure 1). In all subsequent studies,
100 μmol/L homocysteine and 10 μmol/L copper chloride were used, as this
combination elicited maximal effects. Incubation of CVSMCs
with 100 μmol/L homocysteine and
10 μmol/L copper chloride with diphenylene iodonium chloride (DPI), apocynin,
or allopurinol for 16 h reversed
O2·- production (Figure 2),
indicating that the source of
O2·- is NADPH oxidase, by
xanthine oxidase. Co-incubation with SOD
(500 U/mL), catalase (100 U/mL) and sildenafil citrate
(1 μmol/L) also completely inhibited formation of
O2·- in CVSMCs induced with
100 μmol/L homocysteine and 10 μmol/L copper
chloride (Figures 2, 3). Penicillamine inhibited the
formation of O2·- induced by
16-h incubation with 100 μmol/L homocysteine and
10 μmol/L copper chloride (Figure 4). PDE5 expression in rabbit CVSMCs was increased by
100 μmol/L homocysteine and 10 μmol/L copper
chloride, compared to control, following 16 h
incubation (Figures 5, 6). This effect was inhibited by
co-incubation with SOD, catalase, allopurinol, apocynin, and
sildenafil citrate (Figure 5). Co-incubation of
100 μmol/L homocysteine and 10 μmol/L copper chloride with
penicillamine for 16 h reduced PDE5 expression (Figure 6).
4 Discussion
The present study shows that homocysteine alone
or copper alone at (patho)physiological concentrations
have no significant effect on O2·-
formation in rabbit cavernosal smooth muscle cells, following
16-h incubation. Plasma concentrations of copper are between 10
mmol/L and 20 μmol/L [23]. A plasma concentration of
homocysteine above 14.5 μmol/L is considered a risk factor for
vasculopathy [24]. By contrast, homocysteine and copper, in combination, markedly augment the
formation of O2·-. These effects were inhibited by apocynin
and DPI, specific inhibitors of NADPH oxidase, and by
allupurinol, a xanthine oxidase inhibitor [21_22, 25],
after incubation, indicating that the inducible sources of
O2·- are both NADPH oxidase and xanthine oxidase.
Co-incubation of homocysteine and copper with SOD or
catalase over the 16-h incubation phase significantly
reduced O2·- formation, indicating that the endogenous
formation of O2·- and
H2O2 mediates this upregulation of
NADPH oxidase and xanthine oxidase. This is
consistent with a recent study in pulmonary endothelial cells in
which O2·- was shown to directly augment the
expression of NADPH oxidase, thereby generating even more
O2·- through a positive feedback loop [26].
In a previous study, we showed that nicotine and
tumor necrosis factor-α promoted the expression of
PDE5 through an a priori augmentation of NADPH
oxidase and O2·- formation [17]. The present study also
shows that homocysteine + copper together elicited an
increase in PDE5 protein expression in cavernosal cells
following 16-h incubation. This PDE5 expression was
blocked by the co-incubation of cells with SOD, catalase,
and apocynin, indicating that the effect is mediated through
both O2·- and
H2O2, the source of which is NADPH
oxidase. As PDE5 hydrolyses cGMP to inactive GMP,
and cGMP mediates NO-elicited erection, these data
indicate that homocysteine and copper together, but not
alone, might contribute to ED through an upregulation of
PDE5. Although increased PDE5 activity has yet to be
shown in cavernosal tissue in risk factor models for ED,
it has been shown that both cGMP and cyclic adenosine
monophosphate (cAMP) are reduced in the cavernosal
tissue from diabetic animals [27, 28]. As DM is a major
risk factor for ED [29], this indicates that there is an
increase in PDE5 and possibly PDE4 activity in
conditions associated with ED. In the context of the present
study and DM, homocysteine has been shown to augment
O2·- formation from arterial tissue of diabetic
rabbits [30]. Due to the enormous complexity of the
possible systems involved, the intracellular mechanisms
underlying the effect of
O2·- on PDE5 expression was not
addressed in this study. However, possible candidates
include activation of the kinases that modulate
transcription and translation of proteins, including rho and
tyrosine kinases, as well as PKG itself. This area warrants
further study.
Penicillamine, a copper chelator, inhibited the
upregulation of O2·- formation and PDE5 expression.
However, the concentrations at which this was effective
were well below the levels required to chelate copper.
This indicates that penicillamine is exerting an antioxidative
action independently of copper chelation. As penicillamine
reverses ED in the hyperhomocysteinemic rabbit [9], these
other possible mechanisms warrant further investigation.
Several in vitro studies have indicated that
homocysteine alone has little effect on NO formation and mmol/L
concentrations need to be used to elicit inhibitory effects
[18, 19, 31_34]. Even 1 mmol/L is significantly higher
than the concentrations of homocysteine seen even in
severe HHC, which range from 15 μmol/L to
50 μmol/L [24]. The present study consolidates that homocysteine
alone is not an independent risk factor for ED, as 1
mmol/L homocysteine had little effect on
O2·- and PDE5 expression.
By contrast, there are several reports that physiological
concentrations of copper interact with physiological
concentrations of homocysteine to reduce NO formation,
an effect that is dependent on the formation of
O2·- [19, 20]. Under normal circumstances, copper is tightly bound
to plasma proteins, in particular, ceruloplasmin [31].
However, O2·-,
as well as homocysteine itself, dissociates copper from its protein binding sites on
ceruloplasmin and fragments the protein [31], which might
augment the powerful pro-oxidant effects of copper and its
interaction with copper [31]. Furthermore, both
homocysteine and copper alone augment oxidative stress
in arteries from diabetic rabbits but have no effect on
arteries from non-diabetic controls [30, 35].
The present study also indicates that sildenafil citrate,
at therapeutic circulating levels of the drug [11],
significantly reduces PDE5 expression in response to
homocysteine plus copper. Sildenafil also inhibits the activity and
expression of NADPH oxidase in cavernosal and arterial
endothelial cells, through augmentation of the
cGMP_PKG axis [7_11]. Because O2·-
derived from NADPH oxidase upregulates PDE5, we suggest that sildenafil
suppresses PDE5 expression through an a
priori inhibition of NADPH oxidase. This would restore the
NO_cGMP pathway, improving the erectile response. PDE5
inhibitors also potentiate endogenous increases in cGMP
by inhibiting its breakdown at the catalytic site.
Phosphorylation of PDE5 increases its enzymatic activity as
well as the affinity of its allosteric sites for cGMP.
Binding of cGMP to the allosteric site further stimulates
enzymatic activity [36, 37]. Thus, phosphorylation of
PDE5 and binding of cGMP to the noncatalytic sites
mediates negative feedback regulation of the cGMP pathway. Binding of substrate or substrate analogs, such
as tadalafil and vardenafil, to the catalytic site converts a
fast (low-affinity) inhibitor dissociation component of
the PDE5 catalytic site to a slow (high-affinity) inhibitor
dissociation component. This effect is predicted to
improve the substrate affinity or inhibitory potencies of these
compounds in intact cells [36, 37].
From a therapeutic perspective, the present data point
to reducing homocysteine_copper interactions as a means
of improving erectile function. Penicillamine is used to
treat Wilsons's disease, systemic sclerosis, cysteine
stones, and intractable rheumatoid arthritis [38, 39].
However, due to side-effects, it is unlikely that
penicillamine would be an advisable treatment in patients with
ED. In contrast, the folic acid reliably reduces plasma
homocysteine and improves endothelium-dependent relaxation, not only in HHC patients but also in patients
with DM, dyslipidemia, and hypertension [40], all risk
factors for ED. It is reasonable to suggest, therefore,
that folic acid would improve erectile function in patients
with ED. Importantly, there is now a school of thought
that the therapeutic benefit of folic acid is independent of
its homocysteine-lowering effect and that folate and its
active metabolite, 5-methyltetrahydrofolate exerts direct
protective effects on blood vessels that include a
reduction of O2·- formation and prevention of
endothelial nitric oxide synthase uncoupling [41]. Furthermore, the use
of NO donors, which augment cGMP formation, is also
a possible therapeutic strategy for treating ED in pateints
who do not respond favourably to PDE5 inhibitors [42].
In conclusion, this study shows that homocysteine
and copper interact to induce O2·-
formation from NADPH oxidase in rabbit CVSMCs that, in turn, leads to an
upregulation of PDE5. Both O2·-
formation and PDE5 upregulation would impair normal erectile responses.
Sildenafil blocks both of these events, indicating that
prolonged use of sildenafil might be beneficial in treating ED
through inhibition of intra-penile oxidative stress.
Although long-term effects of PDE5 inhibitors on erectile
function in man are not yet published, recent studies have
indicated that long-term therapy with PDE5 inhibitors
improves erectile function in laboratory animals.
Therapeutically, the co-administration of folic acid might
improve the therapeutic impact of PDE5 inhibitors in
patients who do not respond favourably, as they have a
common site of action at the tissue level. Clinical studies
with folic acid are therefore warranted.
Acknowledgment
We thank the Ralph Shackman Trust for financial
support.
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