Endothelial Dysfunction in Erectile Dysfunction: Role of the

21 Pages·2003·1.39 MB·English

JournalofAndrology,Vol.24,No.6Supplement,November/December2003
CopyrightqAmericanSocietyofAndrology
Endothelial Dysfunction in Erectile
Dysfunction: Role of the Endothelium
in Erectile Physiology and Disease
TRINITY J. BIVALACQUA,*† MUSTAFA F. USTA,* in ED. Aging is most commonly associated with ED,but
HUNTER C. CHAMPION,§ PHILIP J. KADOWITZ,† a number of underlying disease processes are recognized
AND WAYNE J. G. HELLSTROM* to lead to abnormal function and responsiveness of the
penile vascular bed.
From the Departments of *Urology and
ED was believed to be a psychological condition; how-
†Pharmacology, Tulane University School of Medicine,
ever,inthepast2decades,authoritieshaverecognizedthat
New Orleans, Louisiana; and the §Department of
the majority of patients’ erectile failure can be attributed
Medicine, Division of Cardiology, Johns Hopkins
to an organic etiology. ED may result from neurologic,
Hospital, Baltimore, Maryland.
arteriogenic, veno-occlusive, or cavernosal impairments
and is therefore associated with vascular risk factors such
Erectile dysfunction (ED) is defined as the consistent in- as atherosclerosis, hypertension, hypercholesterolemia, di-
ability to obtain or maintain an erection for satisfactory abetes mellitus, and cigarette smoking (Feldman et al,
sexual intercourse. Basic science research on erectile 1994;Laumannetal,1999;McKinlay,2000).BecauseED
physiology has been devoted to investigating the patho- ishighlyprevalentinmenwithcardiovasculardisease,and
genesis of ED and has led to the conclusion that ED is because cardiovascular disease is well known to be asso-
predominately a diseaseofvascularorigin.Theincidence ciated with endothelial dysfunction, one can infer that en-
of ED dramatically increases in men with diabetes mel- dothelial dysfunction of the penile vascular tree may con-
litus, hypercholesterolemia, and cardiovascular disease. tributetoimpairmentsinerectilefunction.Therefore,ithas
Loss of the functional integrity of the endothelium and been hypothesized that endothelial dysfunction can result
subsequent endothelial dysfunction plays an integral role
in ED (Maas et al, 2002; Solomon et al, 2003). Recent
in the occurrence of ED in this cohort of men. Thiscom-
clinicalandbasicscienceinvestigationsonaging,diabetes,
munication reviews the role of the vascular endothelium
hypercholesterolemia, and hypertension have shown that
in erectile physiology and the influence of endothelial
endothelial dysfunction is a major contributing factor to
dysfunction in the pathogenesis of ED. Future pharma-
penile vascular pathology.
cological and gene therapy interventions to restore en-
Thefollowingreviewexaminestheroleofthevascular
dothelial function may represent excitingnewtherapeutic
endothelium in erectile physiology and demonstrates the
strategies for the treatment of ED.
importance of the endothelium in normal erectile physi-
Penileerectionisaneurovascularphenomenonthatde-
ology and how impairments in endothelial function can
pends upon neuralintegrity,afunctionalvascularsystem,
cause deleterious effects on erectile function.
andhealthycavernosaltissues(Giulianoetal,1995).Nor-
mal erectile function involves 3 synergistic and simulta-
EndothelialCellFunctionandDysfunction
neous processes: 1) neurologically mediated increase in
penile arterial inflow, 2) relaxation of cavernosal smooth The vascular endothelium not only serves as a passive
muscle, and 3) restriction of venous outflow from thepe- barrier for the arterial and venous blood, but also plays a
nis. The corpus cavernosum of the penis is composed of pivotal role in modulating vascular tone and blood flow
a meshwork of interconnected smooth muscle cells lined in response to humoral, neural, and mechanical stimuli.
by vascular endothelium. Of note, endothelial cells and Furchgott and Zawadzki (1980) first reported the obliga-
underlying smooth muscle also line the small resistance tory role of the endothelium in regulating local andbasal
helicine arteries that supply blood to the corpus caver- control of vessel tone. It is now widely accepted that the
nosum during penile tumescence. Pathological alteration vascular endothelium has a fundamental role in the reg-
in the anatomy of the penile vasculature or impairment ulation of vascular tone in the circulation by releasing a
of any combination of neurovascular processes can result varietyoffactorsthataffectthecontractileandrelaxatory
behavior of the underlying vascular smooth muscle. The
actions of the endothelium are not limited to regulating
Correspondenceto:WayneJ.G.Hellstrom,MD,ProfessorofUrology,
vascular tone, rather, they also play a pivotal role in the
TulaneUniversitySchoolofMedicine,DepartmentofUrology,1430Tu-
laneAveSL42,NewOrleans,LA70112(e-mail:[email protected]). regulation of inflammation, platelet aggregation, vascular
S17
S18 Journalof Andrology · November/December2003
smooth muscle proliferation, and thrombosis (Behrendt addition to NO’s direct toxic effects, it has been shown
and Ganz, 2002). to exhibit an inhibitory effect on smoothmusclecellpro-
The endothelium responds to chemical and hormonal liferation and collagen synthesis.
signals as well as to physical hemodynamic changes TheformationofNOandL-citrullinefromitssubstrate
caused by alterations in blood flow and shear stress by L-arginine occurs in most tissues of the body. The en-
releasing mediators that modulate the tone of the under- zyme that catalyzes this reaction in cells and neurons is
lying smooth muscle layer. In certain disease states, dis- termed NO synthase (NOS). This enzyme uses reduced
ruption of the functional integrity of the vascular endo- nicotinamide adenine dinucleotide phosphate (NADPH),
thelium plays an integral role in the ability of the endo- flavin adenine dinculeotide, flavin mononucleotide, and
thelium to respond to local hemodynamic changes and tetrahydrobiopterin (BH ) as cofactors and heme as a
4
paracrine and autocrine factors, a condition referred toas prosthetic group. The constitutive forms of the enzyme,
endothelial dysfunction (Cai and Harrison, 2000; Beh- neuronal NOS (nNOS; NOS1) and eNOS, are coupled to
rendt and Ganz, 2002; Maxwell, 2002). The regulatory Ca21 and calmodulin and are the principal NOS isoforms
role of the endothelium becomes attenuated during en- involvedintheinductionofpenileerection(Ignarroetal,
dothelial dysfunction, whereby there is either a decrease 1990; Burnett et al, 1992; Rajfer et al, 1992), whereas
in responsiveness to vasodilator mediators or an increase inducibleNOS(iNOS;NOS2)isindependentofCa21and
in sensitivity to vasoconstrictors. Endothelialdysfunction calmodulin and requires new protein synthesis (Alderton
referstoseveralpathologicalconditions,includingaltered et al, 2001). eNOS is predominately membrane bound,
anticoagulationandanti-inflammatoryactivities,impaired whereas nNOS is limited to the cytosol of central and
modulation of vascular growth, and dysregulation ofvas- peripheral neurons, although its mRNA is also localized
cularremodeling(MombouliandVanhoutte,1999).How- to the skeletal muscle (Pollock et al, 1991).
ever, the term endothelial dysfunction is most commonly The concentrations of NO are continuously fluctuating
used to refer to decreases in endothelium-dependent atverylowlevelsthroughoutthevascularsystemandare
smoothmusclerelaxationcausedbyalossoforincreased controlled predominately by eNOS. The constitutive en-
destruction of nitric oxide (NO) bioactivity in the vas- zymes eNOS and nNOS are regulated predominately at
culature. the posttranslational level, whereas iNOS is expressed in
The decrease in NO bioavailability in endothelial dys- response to an appropriate stimulus (eg, cytokines, in-
function may be caused by reductions in the enzyme en- flammation)or transcriptionalfactors(nuclearfactorkap-
dothelialNOsynthase (eNOS,NOS3);alackofsubstrate pa B; NF-kB) (Forstermann and Kleinert, 1995). Recent-
or cofactors for eNOS; alterations in intracellular signal- ly, the subcellular localization of eNOS todistinctmicro-
ing such that eNOS is not appropriately activated or un- domains of the plasmamembrane,itsinteractionwiththe
coupled; or accelerated degradation of NO by reactive protein caveolin-1, and the phosphorylation state of spe-
oxygen species (ROS), such as superoxide anion. Impor- cific serine and threonine residues of the enzyme have
tantly, in endothelial dysfunction, responses to the endo- beenfoundtoplayanintegralpartintheposttranslational
thelium-independent vasodilator sodium nitroprusside regulation of eNOS activity (Feron et al, 1996; Fleming
(SNP) are usually unaltered, indicating that dysfunction and Busse, 1999; Michell et al, 1999; Boo et al, 2002;
arises from abnormal NO production or release from the Goligorsky et al, 2002). NO produced by 1) eNOS from
endothelial cells. However, it is conceivable that other endothelial cells lining the cavernosal smooth muscle
factors, such as superoxide anion, may be preventingNO cells and resistance helicine arteries in response to shear
from eliciting the normal vasodilator response. This con- stress, 2) agonist-induced activation by acetylcholine re-
ditioncausesadisruptioninthebalanceofthevasoactive leased from cholinergic nerves, and 3) nNOS activity in
mediators, whereby the role of vasoconstrictors becomes nonadrenergic, noncholinergic (NANC) neurons is in-
even more prominent and the role of vasodilators dimin- volvedinsignalingeventsthatregulateneurotransmission
ishes, thereby affecting normal vascular tone. and penile vascular tone.
The most important physiological target of NO in the
NO-DependentPenileErection:RoleoftheEndothelium
penis is the heme moiety of soluble guanylate cyclase
Various cellular processes are regulated through the re- (Mizusawa et al, 2002). NO diffuses to adjacent smooth
lease of NO from the endothelium, platelets, vascular muscle cells stimulating guanylate cyclase. This interac-
smooth muscle cells, neurons, and other cell types (Ig- tionconvertsguanosinetriphosphate(GTP)tocyclicgua-
narro et al, 1999). NO has many important physiological nosinemonophosphate(cGMP),whichinducesasubstan-
roles, including neurotransmission, regulation of vascular tial increase in intracellular cGMP, causing smooth mus-
tone, immunomodulation, cell-mediated cytotoxicity cle relaxation. Its action is primarily through the cGMP-
against pathogens and tumor cells, and penile erection dependent kinase I (cGKI, PKG), which alters
(Burnett, 1995; Ignarro et al, 1999; Bogdan, 2001). In intracellularCa21levelsbyreducingCa21channelactivity
Bivalacquaetal · EndothelialDysfunctioninErectileDysfunction S19
and opening Ca21-dependent K1 channels, leading to hy- foreNOShavedemonstrateditspresenceinthetrabecular
perpolarization of the smooth muscle cell (Christ et al, lining of the corpus cavernosum and in the small intra-
1999). PKG can also phosphorylate other proteins to af- cavernosal resistance helicine arteries ofthe penis(Bloch
fect Ca21 channels or lead to an alteration of the phos- et al, 1998; Hedlund et al, 1999; Bivalacqua et al,2001a;
phorylation state of the myosin light chain (MLC) (Mills Gonzalez et al, 2001; Mizusawa et al, 2001; Stanarius et
et al, 2002b). These second messengers reduce intracel- al, 2001). Both human and animal studies have demon-
lular Ca21 via Ca21 sequestration and extrusion and acti- strated that the corpus cavernosum is capable of relaxing
vation of MLC phosphatases. The NO/cGMP-dependent in the presence of endothelium-dependent agonists (ace-
smooth muscle relaxation results in entry of blood and tylcholine, carbachol, bradykinin) and impairment of en-
engorgement of the corpus cavernosum and penile erec- dothelium-dependent cavernosal smooth muscle relaxa-
tion. The physiological actions of cGMP are terminated tion in vitro occurs in vascular-associated diseases, such
by the hydrolysis of the 3959 bond by the type 5 phos- as diabetes mellitus, hypertension, and hypercholesterol-
phodiesterase.TheimportanceoftheproteinkinasecGKI emia (Saenz de Tejada et al, 1989; Gur et al, 2000; Behr-
in the erectile process was established in cGKI-deficient Roussel et al, 2002, 2003). However, what role does en-
mice (Hedlund et al, 2000a). These knockout mice are dothelial-derived NO playintheregulationofnormalpe-
unabletoreproduceandhaveimpairedcavernosalsmooth nile erection in vivo?
muscle relaxation in responsetoneuronalandendothelial In the past 5 years there has been significantevidence
derived NO and exogenous NO. supporting the vital role of endothelial-derivedNOfrom
Until recently, the defined role of eNOS and nNOS in eNOSintheregulationofpenileerectionbothinnormal
the regulation of NO-dependent penile erection has been physiology and in pathological disease states. Our cur-
a subjectof great debate.Micelackingthegenesforboth rent understanding on the mechanism for initiation and
eNOSandnNOSwerestillabletoexhibitnormalerectile maintenance of penile erection is that penile erection is
function and mating behavior (Burnett et al,1996,2002). elicited by neural signals form the spinal cord, which
The first explanation proposed forthemaintenanceofthe stimulates nNOS activity and increases the production
NO-dependent erectile response in nNOS-alpha -/- mice of NO from NANC nerves, thereby causing an increase
was a compensatory up-regulation of eNOS to fulfill in- in blood flow to the cavernosal tissue (Moreland et al,
sufficient nNOS expression. A more recent explanation 2001b). eNOS is then activated by a shear stress/me-
for the intact NO-dependent erectile response in these chanical mechanism by increased blood flow from the
mice is the existence of nNOS gene variants resulting arteries supplying the corpora and expansion of the si-
from alternative mRNA splicing of the nNOS-beta and nusoidal spaces of the corpora. The continued shear
nNOS-gamma alternative translation in exon 1 (Burnett, stress on the endothelial lining of the intracavernosal
2000;Gonzalez-Cadavidetal,2000).eNOS-/-micedem- smooth muscle cells and arteries continues to produce
onstrate normal erectile function to electrical stimulation endothelial-derived NO, which maintains the tumes-
ofthecavernosalnerveevenwhentheirpeniseshaveonly cence phase of penile erection (Figure 1).
60% of the NOS activity compared with wild-type mice Hurt and colleagues (2002), using selective pharma-
(Burnett et al, 2002). Of particular interest, studies from cological inhibitors and eNOS knockout mice, first
eNOS -/- mice have shown direct physiologic evidence showed that penile erection-dependent processes to cav-
for the contribution of eNOS in mediating cholinergic- ernosal nerve stimulation and drug-induced relaxation of
stimulated penile erections, thus documenting the impor- the corpus cavernosum are mediated by phosphatidyli-
tance of cholinergic stimulation and agonist-induced ac- nositol 3-kinase (PI3-kinase) and activation of the serine/
tivation of the endothelium in cavernosal smooth muscle threonine protein kinase Akt. This pathway phosphory-
relaxation and erection (Burnett et al, 2002). Thus, trans- lates eNOS to increase endothelial-derived NO (Michell
genic mice studies with targeted deletion of the eNOS etal,1999).TheuseofpharmacologicalinhibitorsofPI3-
and nNOS genes have further elucidated the role ofthese kinaseinthepenisofratsandeNOS-/-micedemonstrat-
NOS isoforms on the regulation of penile erection and ed that these inhibitors were able to reduce erections to
have reinforced our current understanding of the impor- electrical nerve stimulation and intracavernous papaver-
tance of NO regulatory control of penile erection. ine. This signaling pathway wasfurthermoreshowntobe
NO production from nNOS in the NANC nerves in- responsibleforsustainedNOproductionviaaPI3kinase/
nervating the penis is essential for the initiation of cav- Akt-dependent activation of eNOS with subsequent in-
ernosal smooth muscle relaxation and subsequent erec- creases in endothelial-derived NO and maintenance of
tion. The relative importance of endothelial-derived NO maximal erection (Figure 1).
from eNOS in the endothelial cells of the corpus caver- The application of targeted genes involved in the erec-
nosum and arteries supplying the penis has recently been tile process has further enabled researchers to study the
elucidated. Immunohistochemical and molecular probes mechanisms involved in the pathophysiology of ED-as-
S20 Journalof Andrology · November/December2003
Figure1. SchematicdiagramoftheindependentrolesofnNOSandeNOSintheinitiationandmaintenanceoftheerectileresponse.(Adaptedfrom
Hurtetal,2002.)
sociated conditions, such as aging, diabetes, and hyper- that this NOS isoform was capable of restoring dimin-
cholesterolemia. Both the natural aging process and dia- ished erectile function in aged rats and rats with diabetes
betescausesignificantimpairmentinerectilefunctionthat independent of nNOS expression (Champion et al, 1999;
is contributed to numerous factors both in thecentraland Bivalacquaetal,2000b;BivalacquaandHellstrom,2001;
in the peripheral nervous system as well as at the end Bivalacqua et al, 2003b, in press). These studies have
organ. Most notably, changes in neural integrity of the demonstrated that in disease states, in which eNOS ex-
cavernosal nerve and pelvic plexus as well as in endo- pression is reduced or unaltered, overexpression of this
thelial cell function are well recognized. ED associated NOS isoform is capable of restoring erectile function to
with these conditions is multi-factorial, but most author- cavernosalnervestimulationviaincreasedendothelial-de-
ities recognize that there is an overall reduction in NO rived NO biosynthesis and cavernosal cGMP levels. Im-
biosynthesis(Sullivanetal,1999;Maasetal,2002).Until portantly,eNOSgenetherapyhasnoeffectonnNOSpro-
recently, the independent role of eNOS in these disease tein or gene expression, in that restoration of erectile
processes was unknown. By using adenoviral gene trans- function is solely dependent upon eNOS expression and
ferofeNOStothecorpuscavernosum,itwasdetermined endothelial-derived NO bioactivity. These studies docu-
Bivalacquaetal · EndothelialDysfunctioninErectileDysfunction S21
ment the importance of eNOS in the maintenance of the der the influence of agonists, such as acetylcholine from
erectile response and its significance in pathological con- cholinergic neurons in the penis, and mechanical forces,
ditions associated with ED and endothelial dysfunction. such as shear stress caused by pulsital blood flow in the
sinusoidal spaces of the corpus cavernosum (Hedlund et
Endothelium-DerivedVasodilatorsandVasoconstrictors
al, 2000b). NO diffuses to the underlying smooth muscle
inthePenis
cells where it activates the soluble form of guanylate cy-
A number of review articles address the mechanismsand clase elevating intracellular levels of cGMP and the ac-
pharmacologyofpenileerection(Bivalacquaetal,2000a; tivity of cGKI protein kinase. The NO/cGMP-signaling
Christ, 2000; Andersson, 2001b, 2003). The following cascade reduces contractile activity and promotes caver-
section contains an overview of the endothelium-derived nosal smooth muscle relaxation (see previous section).
vasodilator and vasoconstrictor agents involved in the Prostaglandin—NO can activate prostaglandin (PGE)
physiology of penile erection. synthesis in vivo. During shear stress blood flow in the
Cavernosal smooth muscle cells in the penis are pre- penis,anothermechanisminvolvedtoenhancecavernosal
dominately found in the contracted state with minimal smooth muscle relaxation is activation of PGE synthesis
bloodflowingthroughthecavernoussinuses.Thebalance viaaNO-dependentmechanism(Ballermannetal,1998).
between known contractile systems (RhoA/Rho-kinase, The vasodilators PGE and PGI are primarily produced
2
a-adrenergic, endothelin, angiotensin, thromboxane A ) by endothelial cells in the vasculature. PGI is the most
2 2
and vasodilatory second-messenger systems (adenylate abundant arachidonic acid product generated in vascular
cyclase-cyclic AMP and guanylate cyclase-cyclic GMP) tissues (McNamara et al, 1998). Both isoforms of cyclo-
determines the overall tone of corpora cavernosa smooth oxygenase, COX-1 and COX-2, can convert arachidonic
muscleofthepenis(Andersson,2001a;Mills,2002).This acidtoPGH .PGH issubsequentlyconvertedtoPGI by
2 2 2
balanceiscontrolledbybothcentralandperipheralmech- the action of PGI synthase (PGIS) and PGE by PGE
2 2 2
anismsandinvolvesaplethoraofneurotransmittersacting synthase.PGEsynthesizedbyendothelialcellsinthecor-
through various signal transduction pathways. pus cavernosum in response to mechanical shear stress
Endothelial cells actively regulate basal vascular tone blood flow in the penis binds to specific PGE (EP) re-
and vascular reactivity in physiological and pathological ceptorsonsmoothmusclecells(Traishetal,1997;Megh-
conditions by responding to mechanical forces and neu- dadi et al, 1999). Activation of EP receptors by PGE in-
rohumoral mediators with the release of a variety of re- creases intracellular levels of cAMP via activation of ad-
laxing and contracting factors. In the corpuscavernosum, enylate cyclase, causingareductioninintracellularlevels
the vascular endothelium and cavernosal arteries are a of Ca21 and cavernosal smooth muscle relaxation (More-
source of vasorelaxing factors such as NO; prostacyclin land et al, 2001a). Both PGE and itsderivativePGE are
2 1
(PGI ); the not-yet identified endothelium-derived hyper- potent vasorelaxing agents in human corpus cavernosum
2
polarizing factor (EDHF); and the vasoconstrictor factors smooth muscle. There are 4 distinct EP receptors(EP ),
1–4
angiotensin II (Ang II), endothelin-1 (ET-1), and Rho- andall4areexpressedinthecorpuscavernosum(Angulo
kinase. These endothelium-derived factors have a regu- etal,2002).EP andEP areG-protein–coupledreceptors
2 4
latory influence on cavernosal vascular tone. Normally, and are responsible for an increase in cAMP synthesis in
these factors act in concert to elicit an overall beneficial response to exogenous PGE administrationtothehuman
1
effect on cavernosal smooth muscle function, which is penis and in cultured cavernosal smooth muscle cells.
crucial in response to changes in blood flow,shearstress, PGE is a potent vasodilator of the penile vascular bed
1
and agonists in order to maintain physiological homeo- and is a highly efficacious local agent for the treatment
stasisthroughoutthepenilevascularbed.Thecriticalbal- of ED (Leungwattanakij et al, 2001).
ance of vasodilators and vasoconstrictors is normally EDHF—The currently unidentified EDHF likely plays
maintainedduringhealthandquicklyrespondstochanges animportantroleinerectilephysiology.EDHFisreleased
in blood flow and other factors. Decreases in NO-, pros- by endothelium-dependent agonists or shear stress and
taglandin-, and EDHF-mediated responses have been hyperpolarizes the underlying smooth-muscle–inducing
shown to be involved in cardiovascular diseases related relaxation by decreasing intracellularlevelsofCa21with-
to endothelial dysfunction, and increases in responses to in the smooth muscle cells (Busse et al, 2002). This
Ang II and ET-1 have also been implicated (Harrison, mechanism involves decreasing the opening probability
1997;DeVrieseetal,2000).Evidencenowexiststodem- of the voltage-dependent Ca21 channels and reducing the
onstrate that this also occurs in the penile vascular bed. turnover of intracellular phosphotidylinositides. EDHF-
NO—The principle mediator of cavernosal smooth mediated responses increase as the vessel size decreases
muscle relaxation is NO released by the NANC neurons (Mombouli and Vanhoutte, 1997). It was once believed
innervating the penis and by cavernosal endothelial cells that the final common pathway leading to smoothmuscle
(Burnett,1995).NOisreleasedfromendothelialcellsun- hyperpolarization by EDHF required the opening of po-
S22 Journalof Andrology · November/December2003
tassium channels on the smooth muscle cell membrane;
however, this hypothesis has been modified in light of
more recent experimental observations.
Current evidence suggests that EDHF is formed after
an increase in endothelial Ca21, either induced by an ag-
onist (acetylcholine, bradykinin) or shear stress that trig-
gers the synthesis of a cytochrome P450 metabolite,
which is essential for the subsequent EDHF-mediatedre-
sponse (Fisslthaler et al, 1999; Fleming, 2001). Epoxyei-
costatrienoic acid (EETs) is an arachidonic-acid–derived
product of cytochrome P450 epoxygenases that appears
to play an important role in the regulation of vascular
homeostasis(Fleming,2001;Zhangetal,2001).EETsare
reported to be potent vasodilators in a number of periph-
eral vascular beds. In human coronary arteries,inhibitors
ofcytochromeP4502CblockEDHF-mediatedresponses, Figure2. Bargraphdepictingthevoltage-dependenterectileresponse
(ICP/MAP) after cavernosal nerve stimulation at the 5 V setting for 1
and EETs relax coronary arteries by hyperpolarizing
minuteincontrolratsbeforeandafteradministrationofthecytochrome
smooth muscle cells through a K-channel–dependent P450 2C inhibitor, sulfaphenazole, in a dose of 100 mg/kg IV. The in
mechanism (Miura and Gutterman, 1998; Miura et al, vivoerectileresponsewasmeasuredatbaselineand30minutesafter
administrationofsulfaphenazole.nindicatesnumberofexperiments;*,
1999). However, the role of EDHF-mediated cavernosal (P,.05)responsesignificantlydifferentfrombaselineresponse.
smooth muscle relaxation has not been determined until
recently. Angulo et al (2003) revealed that an EDHF-me-
diated relaxation of human penile resistance arteries ex- tide generated in the vascular endothelium that is recog-
ists, which is resistant to NOS and COX inhibition, sug- nized as a potent and sustained vasoconstrictor in the pe-
gesting that EDHF may play an important role in the en- nile vasculature (Christ et al, 1995; Mills et al, 2001b).
dothelium-dependent relaxation of the penile vascular The effects of ET-1 are mediated through ET receptors,
A
bed. Additionally, our laboratory has found that thephar- which are located on the underlying smooth muscle, and
macological inhibitor ofcytochromeP4502C,sulfaphen- through ET receptors, which are located on the smooth
B
azole, attenuates cavernosal nerve mediated erectile re- muscle and vascular endothelium. ET receptors mediate
A
sponses in the rat. This suggests that a cytochrome P450 contraction and promote growth of smooth muscle. ET
B
metabolite may mediate an EDHF-dependent smooth receptors on smooth muscle also mediate contractions,
muscleeffectinthepenisthatmaycontributetotheerec- whereas stimulation of ET receptors on the endothelium
B
tile response (Figure 2). Because this area of penile vas- promote NO and prostacyclin-mediated vasorelaxation.
cularbiologyisnotfullyelucidated,furtherresearchmust Of note, ET-1 acts as a vasodilator at low doses (ET
B
be undertaken to evaluatethepotentialimportanceofthis receptoractivation)andasavasoconstrictor(ET receptor
A
endothelium-derivedrelaxingfactorintheregulatorycon- activation)athighdoseswhenadministeredtocavernosal
trol of penile erection. strips in vitro. Basal production of ET-1 by endothelial
Ang II—Ang II is a potent vasoconstrictor. In addition cells of the corpus cavernosum is hypothesized to con-
to the classical renin-angiotensinsystem(RAS)operating tributetosustainedcavernosalsmoothmusclecontraction
systemically,thereisafunctionalRASthatgeneratesAng andmaintenanceofpenileflaccidity.Bothanimalandhu-
II locally in the vascular tissue of the penis (Kifor et al, man studies have not validated this hypothesis; therefore,
1997). Angiotensin receptors characterized in the penile despite the existence of ET receptors in the penis, most
vasculature suggest that endothelial cells in the corpus whole animal experimental and human clinical data do
cavernosummayformalocalAngII–producingparacrine not support a central role for ET-1 in regulating the nor-
system that may modulate vascular tone by keeping the mal erectile response (Becker et al, 2000, 2001b; Dai et
cavernosal smooth muscle cells in a constricted state al, 2000; Kim et al, 2002). Diabetic corpus cavernosum
(Park et al, 1997). In organ bath studies of isolated strips obtained from animal models and humans have shown
of corpus cavernosum, Ang II caused a dose-dependent that ET receptors are upregulated and ET receptors are
A B
contraction of the cavernosal tissue in vitro (Beckeretal, downregulated, suggesting that ET-1 may be involved in
2001c).ElevatedlevelsofAngIIhavebeennotedinmen the pathogenesis of diabetic ED (Sullivan et al, 1997,
with organic ED, suggesting this peptide may play a role 1998; Chang et al, 2003). However, future studies are
in the pathogenesis of ED (Becker et al, 2001a). warranted to establish the functional significanceofthese
ET-1—The endothelins (ET-1, ET-2, and ET-3) are a cavernosal molecular changes in the pathophysiology of
family of related peptides. ET-1 is a 21 amino acid pep- diabetic-associated endothelial dysfunction.
Bivalacquaetal · EndothelialDysfunctioninErectileDysfunction S23
arginase isoforms have been localized in the human cor-
pus cavernosum (Bivalacqua et al, 2001b). Moreover, in-
hibition of arginase with 2(S)-amino-6-boronohexanoic
acid (ABH) is associated with enhanced NANC- and en-
dothelium-dependent vasorelaxation of human corpus
cavernosum smooth muscle, suggesting that inhibition of
arginase will increase NO biosynthesis through a NOS-
dependent manner (Cox et al, 1999). Theoretically, it is
feasible to regulate NO biosynthesis in both endothelial
and smooth muscle cells of the penis by controlling the
availability of arginine to react with NOS, andregulation
of the arginase enzyme can accomplishthis.Arginaseac-
tivityhasbeenshowntobeupregulatedindiabetichuman
corpus cavernosum, suggesting that the diminished erec-
tile response caused by decreased NO production found
in men with diabetes may be due to a combination of
Figure 3. Arginine metabolism. L-arginine enters the endothelial cell
through cationic amino acid transporter (CAT) and can react with the increased expression of arginase and decreased amounts
enzymesarginaseoreNOS.WhenL-argininereactswitheNOS,NOand of NOS nerve fibers (see section on Endothelial Dys-
L-citrullineisformed.NOcanthenbindtothesolubleformofguanylate
function and Diabetes). Additionally, the expression of
cyclase(sGC)toformcGMP.WhenL-argininereactswitharginase,L-
ornithineandureaareformed.Ornithinecanreactwithornithinedecar- CAT transporters is also a potential determinant of the
boxylase(ODC)toformpolyaminesthatcontributetocellproliferationor rate of L-arginine delivery to eNOS and thus can be an-
reactwithornithineaminotransferase(OAT)toformpyrroline-5-carbox-
other rate-limiting step in NO biosynthesis. Factors that
ylate(P5C).Ureacanformreactiveoxygenspecices(ROS)insomecell
types. affectCATactivityandtherateoftransportofL-arginine
inendothelialcellsincludetheconcentrationofothercat-
ionic amino acids, oxidized lipoproteins, glucose, and in-
Arginase—Arginine is a precursor for the synthesis of sulin (Zharikov and Block, 1998). Thus,diabetesandhy-
NO, urea, polyamines, creatine phosphate, and various perlipidemiamayinfluenceargininetransportinthepenis
proteins (Figure 3). This amino acid is transported from and contribute to endothelial dysfunction observed in
the circulation into mammalian cells by cationic amino these pathological states.
acidtransporter(CAT)isoforms(Durante,2001).Cationic RhoA/Rho-kinase—Although the mechanisms involved
amino acids such as L-arginine are transported into cells in the regulation of the vasorelaxation of the penile vas-
via the y1 transport system. This systems activity is me- cularbedbyendothelial-derivedvasodilatorshasbeenex-
diated by the CAT family, which is composed of 4 iso- tensively studied, the endothelial-derived agentsinvolved
forms: CAT-1, CAT-2A, CAT-2B, and CAT-3. The exis- in the maintenance of the contracted state are only re-
tenceoftheseCATtransportershasnotbeendocumented cently gaining widespread attention. Contraction of cav-
in the penis, but their existence is inevitable. The major ernosalsmoothmuscleisprimarilymediatedbytheCa21-
site of arginine metabolism is the liver, where L-arginine dependent activation of MLC kinase, resulting in the
generated in the urea cycle is converted to urea and or- phosphorylation of MLC, and subsequent actin/myosin
nithine by the enzyme arginase. Many additional tissues cross-bridge formation (Figure 4). In addition, recent ev-
and cell types also contain the enzyme arginase, in par- idence has established the important role of Ca21-sensi-
ticular endothelial cells (Li et al, 2002). tization through theCa21-independentpromotionofMLC
In endothelial cells, arginine is used as a substrate by kinase or the attenuation of MLC phosphatase activity
botheNOSandarginase.BecausebothNOSandarginase (Chitaley et al, 2001b, 2003). A principle regulator of
use arginine as a common substrate, arginase may down- MLC phosphatase is the serine/threonine kinase, Rho-ki-
regulate NO biosynthesis by competing with NOS for L- nase. Data from other vascular beds suggest that RhoA,
arginine (Figure 3). Thus, NO production is likely to be a member of the Ras low molecularweightofGTP-bind-
linked to the regulation of arginase activity (Bivalacqua ing proteins, mediates agonist-induced activation of Rho-
et al, 2001b; Kim et al, 2001). Arginase exists in 2 iso- kinase(Wettschureck andOffermanns,2002;Swardetal,
forms: the hepatic type (arginase I) and the extrahepatic 2003). The exchange of GDP for GTP on RhoA and
type (arginase II) (Mori and Gotoh, 2000). Recently, our translocation of RhoA from the cytosol to the membrane
laboratory has shown thatendothelialcellsobtainedfrom are markers of its activation and enable the downstream
the mouse corpus cavernosum contain both arginase iso- stimulation of various effectors such as Rho-kinase. Nu-
forms (Bivalacqua et al, 2002). Additionally, by using merous studies have established an important role for
semiquantitative RT-PCR and Western blot analysis,both RhoAandRho-kinaseinnumerouscellularresponses,in-
S24 Journalof Andrology · November/December2003
enhanced erectile function, suggesting that inhibition of
RhoA/Rho-kinase expression in the penis can augment
cavernosal smooth muscle relaxation and subsequent
erectilefunction(Chitaleyetal,2002).TreatmentwithY-
27632 also potentiated voltage-dependent (NO-mediated)
increases in erectile function, and inhibitors of NOS or
guanylate cyclase did not block this effect (Mills et al,
2002a). Studies by Sauzeau and colleagues (2000, 2003)
have demonstrated that NO and PKG can inhibit the
translocation of RhoA to the membrane in the rat aorta.
Additionally,SNPwasfoundtoreversethephenylephrine
(PE)-induced translocation of RhoA in the rat aorta,
which is further indicative of NO’s inhibitory action on
RhoA activity (Chitaley and Webb, 2002). In the rat pe-
nis, NO mediates the erectile response, in part, via inhi-
bition of the RhoA/Rho-kinase Ca21-sensitizing pathway.
These data demonstrate that NO inhibits Rho-kinase ac-
tivity,supportingthehypothesisthatendogenousNO-me-
Figure 4. RhoA/Rho-kinase signal transduction pathway and its inter-
diated vasodilation may occur through the inhibition of
actionwitheNOSinthepenilevascularbed.RhoAisactivatedbyeither
agonist-induction (norepinephrine, endothelin-1) or vascular disease Rho-kinase vasoconstrictor activity.
states(diabetes,hypertension)whenitbindsGTP,undergoesgeranyl- Recent evidence suggests that the RhoA/Rho-kinase
geranylation,andmigratestothecellmembrane.ActivatedRhoAstim-
signal transduction pathway is an important signal me-
ulatesRho-kinaseexpression,which,inturn,reduceseNOSproteinand
mRNA stability and catalyzes the phosphorylation (andinactivation)of diator of penile vascular endothelial cell function (Biva-
myosin light chain (MLC) phosphatase. This in turns promotes caver- lacqua et al, 2003d). The RhoA/Rho-kinase pathway
nosalsmoothmusclecontractionvia2separatemechanismsofaction.
plays an important role in suppression of eNOS gene ex-
When MLC phosphatase is in the active form (nonphosphorylated),it
catalyzes the dephosphorylation of MLC and thereby promotescaver- pression and enzyme activity in human endothelial cells,
nosal smooth muscle relaxation. This process occurs whenthe down- which results in decreased endothelial-derived NO bio-
streammediatorofthe NO/cGMPsystem,PKG,inhibitsRhoAtranslo-
synthesis (Figure 4). This mechanism may play an im-
cationtothemembraneandreducesRho-kinaseexpression.Thisresults
inanincreaseineNOSexpressionandendothelial-derivedNOandcav- portant role in the regulation of penile endothelial cell
ernosalsmoothmusclerelaxationthrough2separatemechanisms,de- function and dysfunction as related to vascular diseases
phosphorylationofMLCandincreasedeNOSexpression.
ofthepenilevasculature(seesectiononEndothelialDys-
function and Diabetes).
cluding the contraction of smooth muscle cells (Uehata ROS—ROS are a family of molecules produced by all
et al, 1997; Chitaley et al, 2001a). Both norepinephrine aerobic cells. Oxidation of biological molecules (DNA,
(NE) and ET-1 stimulate the GTPase RhoA in vascular protein,lipids)occursasaresultofincreasedsteady-state
smooth muscle cells, suggesting NE and ET-1 may cause levels of ROS or decreased antioxidant defense mecha-
vasoconstriction through a RhoA/Rho-kinase-dependent nisms. This phenomenon is commonly referred to as ox-
manner. Although the role of RhoA/Rho-kinase has been idative stress. Many ROS possess unpaired electrons and
welloutlinedinnumerousformsofsmoothmuscle,recent therefore are free radicals. These include molecules such
evidence has demonstrated its importance in the regula- as superoxide anion, the hydroxyl radical, lipid radicals,
tion of cavernosal smooth muscle tone (Chitaley et al, andNO.Hydrogenperoxideandperoxynitritearenotfree
2001b, 2002; Mills et al, 2001a; Rees et al, 2001). Both radicals by definition but have deleterious oxidative ef-
human endothelial cells and human corpus cavernosum fects that contributetooxidativestressinmanycelltypes
smooth muscle cells grown in culture express RhoA and such as the vascular endothelium. Endothelial cells se-
Rho-kinase (Essler et al, 1998; Rees et al, 2002; Wang et crete ROS in response to shear stress, endothelium-de-
al, 2002; Wojciak-Stothard and Ridley, 2003). pendent agonists (acetylcholine, bradykinin), and in var-
Evidence supporting the role of Rho-kinase in the ious vascular disease states.
maintenance of cavernosal smooth muscle vasoconstric- One of the most widely studied endothelial-derived
tion has been demonstratedbyadministrationoftheRho- ROS in the vasculature is superoxide anion. Superoxide
kinase inhibitor, Y-27632, directly into the cavernosal si- anion is produced in a variety of cells, including neutro-
nuses of rats, which caused dose-dependent increase in phils, monocytes, B-lymphocytes, platelets, mast cells,
intracavernosal pressure (Chitaley et al, 2001b). Addi- vascularsmoothmusclecells,andendothelialcells(Mun-
tionally, adeno-associated viral gene transfer of the dom- zel et al, 1997; Wolin et al, 2002). Several cells, most
inantnegativeRhoAmutanttotheratcorpuscavernosum notably endothelial cells, use the membrane-associated
Bivalacquaetal · EndothelialDysfunctioninErectileDysfunction S25
enzyme, NADH phosphate oxidase (NADH/NADPH),to percholesterolemia. These vascular disorders are highly
generate superoxide anion (Munzel et al, 1999). Other prevalent in patients with ED and have been identifiedas
potential sources of ROS in endothelial cells include li- independentriskfactorsforEDinlarge,population-based
poxygenase and COX (arachidonic acid pathway en- studies.IncreasedinactivationofNObysuperoxideanion
zymes), peroxidases, cytochrome P450s, xanthine oxi- in conditions of increased oxidative stress creates an im-
dase, and eNOS. balance that leads to a deficit of endothelial-derived NO
The reaction of superoxide anion and NO in the vas- acutely and ultimate development ofendothelialdysfunc-
cular endothelium or smooth muscle cells results in the tion. Superoxide production can occur in the vasculature
formation of the highly toxic molecule, peroxynitrite through the actions of 3 major enzyme systems: NADH/
(Wolin, 2000). Peroxynitrite is known to cause tissue in- NADPH oxidases, xanthine oxidase, and eNOS. Besides
jury, alterations in vascular tone, oxidation of cellular reducing NO-biosynthesis, superoxide anion directlypro-
proteins and lipids, apoptosis, and organ dysfunction via motes a number of events, which may lead to impair-
its direct toxic effects (Beckman et al, 1990; Beckman ments in penile erection (Jones et al, 2002). Superoxide
andKoppenol,1996).Thisreactionis30timesfasterthan anion can cause Ca21 mobilization, thus reducing intra-
that of NO and oxyhemoglobin and 3 times faster than cellular levels of Ca21 in the cavernosal endothelialcells,
the dismutation of superoxide-by-superoxide dismutase whereas an increased production of peroxynitrite leadsto
(SOD). Given this rapid reaction rate, there is usually generation of vasoconstricting agents and potential apo-
some superoxide anion reactingwithNOwithincellsand ptosis of endothelial cells. These events may contribute
the extracellular space. The antioxidants SOD, catalase, to impairments in the penile vasculature observed in ED
glutathione peroxidase, and reductase play an important associated with conditions known to increase oxidative
role in the cellular protection against ROS (Kunsch and stress and superoxide anion production.
Medford, 1999). SOD represents a major cellulardefense The majority of experimental data on the role of su-
against superoxide anion and peroxynitrite formation by peroxide anion on corpus cavernosal and endothelial
accelerating the dismutation of superoxide into H O and function in the penis has been studied in animal models.
2 2
molecular oxygen (Fukai et al, 2002). Three SOD iso- The effect of a superoxide anion-generating agent on in
forms have been identified, including a cytosolic copper/ vitro NO-mediated cavernosal smooth muscle relaxation
zinc-containing form (CuZnSOD), a mitochondrial man- demonstrated that acetylcholine-mediated relaxation was
ganeseform(MnSOD),andanextracellularisoform(EC- impaired in the rat corpus cavernosum in the presence of
SOD), which is also a copper/zinc-containing enzyme. increased productionofsuperoxideanion,suggestingthat
Givenitslocation,EC-SODishypothesizedtoplayacrit- superoxide anion can impair endothelial-derived NO in
ical role in modulating the redox state of the vascular normal erectile tissue (Cartledge et al, 2000). However,
interstitium and thereby preventing the pathophysiologi- the interaction of superoxide anion and NO-mediated
cal effects of superoxide anion in the vasculature,specif- erectileresponsesbothinvitroandinvivohasbeenmore
ically the endothelial and smooth muscle cells (Fukai et extensively studied in diseased animal models of ED.
al, 2002). Both the Cu/Zn and EC isoforms have been It is well known that hypercholesterolemia results in
found in the penis, predominately in the endothelial and significant impairments in endothelium-dependent corpus
cavernosalsmoothmusclecells(Bivalacquaetal,2003a). cavernosal smooth muscle relaxation. Rabbits fed high-
Conceptually, an increased level of superoxide anion in cholesterol diets developed impairments in corpus cav-
the endothelium and cavernosal smooth musclemaycon- ernosal endothelial smooth muscle relaxation at a time
tribute to ED by decreasing penile NO biosynthesis and when cavernosal superoxide anion levels were signifi-
causingendothelialdysfunctionascommonlyobservedin cantly elevated in the hypercholesterolemic group (Kim
thevasculareffectsofdiabetes,hypercholesterolemia,and et al, 1997).Additionally,therewasasignificantincrease
aging. This is an important concept because cavernosal in cavernosal total CuZn levels and MnSOD levels, in
smooth muscle relaxation and ultimately penile erection order to scavenge the excess superoxide anion present as
dependuponthesustainedproductionofNObyahealthy a result of hypercholesterolemia. In spite of the elevated
endothelium. SOD levels, there were still significant impairments in
endothelium-dependent smooth muscle relaxation.
EndothelialDysfunctionandOxidativeStressinthe
Low-density lipoprotein (LDL) peroxidation contrib-
Penis
utes tothedevelopmentofatherosclerosis,andinjuriesto
A considerable body of evidence implicates oxidative endothelial cells have a principal role in the progression
stress, in particular the reaction of NO and superoxide of atherosclerotic lesions (Rubbo et al, 2002). Oxidized
anion, as an important pathogenic element in the devel- LDL (ox-LDL) is an oxidative stress-derived pathogen
opment of endothelial dysfunction in vascular diseases formed by superoxide anion and peroxynitrite and highly
such as diabetes, hypertension, arteriosclerosis, and hy- associated with hypercholesterolemiaandatherosclerosis.
S26 Journalof Andrology · November/December2003
Ox-LDL has been shown to impair endothelium-depen- Aging is associated with marked changes in the penis,
dent relaxation in the penis and may also contribute to especially in the endothelium and smooth muscle cells
endothelialdysfunctionobservedinhypercholesterolemia (Harman, 2001; Drew and Leeuwenburgh, 2002). Oxi-
through an increased production of superoxide anion via dative stress is an important factor contributing to vas-
uncoupling of eNOS or a reduction in the eNOS cofactor cular dysfunction in aging (Hamilton et al, 2001; Taddei
BH (Ahn et al, 1999). Additional studies are warranted et al, 2001). Superoxide anion levels are increased in the
4
to delineate the exact mechanisms involved in a super- cardiovascular system and a number of peripheral vas-
oxide anion-dependent mechanism of endothelial dys- cular beds with advancing age. Therefore,age-relateden-
function of the penile vascular bed associated with ath-
dothelial dysfunction of the penis may involve mecha-
erosclerosis and hypercholesterolemia.
nisms such as increased oxidative free radicals or alter-
Numerous animal and human experimental data have
ationsinantioxidantdefensesystems.Agingisassociated
demonstrated that diabetic vasculopathy and neuropathy
with decreased NO-bioavailability and responsiveness to
contribute significantly to diabetic-associated ED. Im-
endothelium-dependent vasodilator stimuli in the corpus
pairments in endothelium-dependent and NANC-mediat-
cavernosum, and the role superoxide anion plays in me-
ed cavernosal smooth muscle relaxation are well estab-
diating this decreased responsiveness had not been estab-
lished in diabetic corpus cavernosum in vitro and in vivo
lished until recently. Because superoxide anion reacts
(Saenz de Tejada et al, 1989; Vernet et al, 1995; Rehman
with NO to form peroxynitrite, this reaction does not al-
et al, 1997; Way and Reid, 1999; Gur et al, 2000; Car-
low NO to perform its role in vasodilation and penile
tledge et al, 2001b; Bivalacqua et al, 2003b). Recent ev-
erection. In the aging penis, overproduction of toxic rad-
idence suggests that oxidative stress may play a promi-
ical peroxynitrite could cause degeneration of nervesand
nent role in diabetic endothelial dysfunction ofthepenile
endothelial cells involved in the erectile process (Ferrini
vascular bed. Endothelium-dependent cavernosal smooth
muscle relaxation is impaired in organ bath studies with et al, 2001; Bivalacqua et al, 2003a). Thus, reduced en-
alloxan-induceddiabeticrabbitcavernosaltissue(Khanet dothelial-derived NO may be a result of increased pro-
al, 2001). SOD treatment restored endothelial- and duction of superoxide anion in the aged penis. Recently,
NANC-mediated corpus cavernosum smooth muscle re- in situ detection of superoxide was shown to be elevated
laxation, demonstrating the functional significance of su- in endothelial and cavernosal smooth muscle cells of the
peroxideanioninmediatingdiabeticendothelialdysfunc- aged rat (Figure 5) (Bivalacqua et al, 2003a). Although
tion in vitro. Ryu et al (2003) have shown that malon- therewasanincreaseinsuperoxideanionformation,there
dialdehyde, a lipid peroxidation productthatisameasure was no change in total SOD activity, such that there was
of oxidative stress, is increased in cavernosal tissue ob- an imbalance in superoxide anion generation and inacti-
tained from streptozotocin (STZ)-induced rats with dia- vation in the penile vasculature of the aged rat. This in-
betes. Our lab has recently found a significant up-regu- crease in superoxide anion generation is associated with
lation of superoxide anion, driven by the membrane- decreased NO synthesis in the penis, thus impairing en-
bound NADH/NADPH oxidase, in cavernosal tissue of
dothelial-dependent smooth muscle relaxation and result-
STZ-induced rats with diabetes with no change in total
inginreducederectilefunction(Bivalacquaetal,2003a).
SOD activity (Bivalacqua et al, unpublished data). These
It was this imbalance in superoxide anion formation and
animals had concomitant ED as measured by cavernosal
SOD activity that led us to hypothesize that increasing
nerve-inducederectileresponsesinvivo.Adenoviralgene
EC-SOD expression might be beneficial in limiting su-
transfer of EC-SOD to the diabetic rat penis reduces the
peroxide anion formation. In rats transfected with Ad-
marked increase in cavernosal superoxide anion levels
CMVEC-SOD, expression of EC-SOD mRNA and pro-
andcompletelyrestoredcavernosalnerve-inducederectile
tein was significantly higher and resulted in a significant
function in this cohort of animals (Bivalacqua et al, un-
reduction in superoxide anion formation in the aged rat
published data). These results suggest that an increase in
penis. Additionally, EC-SOD gene therapy reduced per-
superoxideanionformationcausedbyanup-regulationof
oxynitrite formation and increased cGMP levels in the
NADPH oxidase without a compensatory increase in
penile vasculature that was associated with enhanced in
SOD occurs in the penis of the STZ-induced rat with
diabetes and that gene therapy of EC-SOD can reduce vivo erectile responses to cavernosal nerve stimulation
superoxide anion levels and restore erectile function in and the endothelium-dependent vasodilator acetylcholine
vivo. These studies provide evidence that in diabetes su- in the aged rat (Bivalacqua et al, 2003a). These data in-
peroxide radicals are increased, possibly by the up-regu- dicate a pathophysiologic role for superoxide anion in
lation of NADPH oxidase, and contribute significantly to augmenting erectile function in aging by reducing bio-
endothelial dysfunction of the penile vasculature in dia- availability of NO and impairing endothelial function in
betes. the penis.