International Journal of Impotence Research (2012) 24, 49–60
& 2012 Macmillan Publishers Limited All rights reserved 0955-9930/12
www.nature.com/ijir
REVIEW
The promise of inhibition of smooth muscle tone as a treatment
for erectile dysfunction: where are we now?
X Jiang and K Chitaley
Department of Urology, University of Washington, Seattle, WA, USA
Ten years ago, the inhibition of Rho kinase by intracavernosal injection of Y-27632 was found to
induce an erectile response. This effect did not require activation of nitric oxide-mediated signaling,
introducing a novel target pathway for the treatment of erectile dysfunction (ED), with potential
added benefit in cases where nitric oxide bioavailability is attenuated (and thus phosphodiesterase
type 5 (PDE5) inhibitors are less efficacious). Rho-kinase antagonists are currently being developed
and tested for a wide range of potential uses. The inhibition of this calcium-sensitizing pathway
results in blood vessel relaxation. It is also possible that blockade of additional smooth muscle
contractile signaling mechanisms may have the same effect. In this review, we conducted an
extensive search of pertinent literature using PUBMED. We have outlined the various pathways
involved in the maintenance of penile smooth muscle tone and discussed the current potential
benefit for the pharmacological inhibition of these targets for the treatment of ED.
International Journal of Impotence Research (2012) 24, 49–60; doi:10.1038/ijir.2011.49; published
online 6 October 2011
Keywords: contraction; erectile dysfunction; RhoA/Rho kinase; smooth muscle
Introduction
Erectile dysfunction (ED) affects 30 million men
in the United States1 and is associated with
co-morbidities ranging from prostatectomy to diabetes and increased age. Penile erection is a
dynamic process requiring dilation of feeder arterioles and cavernosal sinusoids allowing for increased inflow of blood. It is important to
remember, however, that the vast majority of the
time, these arterioles and sinusoids are maintained
in the collapsed/contracted state, severely restricting penile blood flow.2 The maintenance of penile
flaccidity through vasomotor tone is an active
process involving complex signaling mechanisms.
Heightened smooth muscle contraction is present in
some models of ED,3,4 and various studies have
suggested that pharmacological inhibition of
smooth muscle contraction, as opposed to active
induction of dilation (such as through nitric oxide
Correspondence: Dr K Chitaley, Department of Urology,
University of Washington, Box 358050, 815 Mercer Street,
Room 319, Seattle, WA 98109, USA.
E-mail: [email protected]
Received 28 March 2011; revised 13 July 2011; accepted
17 August 2011; published online 6 October 2011
(NO)-mediated pathways), may be a beneficial strategy for the treatment of ED.5–7
In this review, we will outline the major smooth
muscle signaling pathways involved in penile
vasoconstriction and discuss the potential for
inhibition of these pathways as a treatment option
for organic ED. A focus will be on the promise
and limitations of pharmacological therapy based on
current progress in the development of RhoA/
Rho-kinase (ROCK) antagonists.
Contractile signaling
The vast majority of time, the penis is maintained in
the flaccid state through active contraction of penile
arterioles. The release of norepinephrine (NE) from
sympathetic nerve terminals activates arteriolar and
cavernosal a-adrenergic receptors.2,8,9 Subsequent
increases in intracellular Ca2 þ concentration ([Ca2 þ ]i)
result in the activation of myosin light-chain kinase
(MLCK) and phosphorylation of myosin light chain
(MLC), enabling actin–myosin cross-bridge cycling.
In addition to the Ca2 þ -dependent contractile
mechanism, Ca2 þ -sensitizing pathways, such as
ROCK- and protein kinase C (PKC)-mediated signaling, can promote contraction through the inhibition
of MLC phosphatase or the direct stimulation of
MLC phosphorylation.10,11 Both Ca2 þ -dependent
Inhibition of smooth muscle tone as a treatment for ED
X Jiang and K Chitaley
50
and Ca2 þ -sensitizing signaling can be activated by
NE or other agonists, including endothelin-1 (ET-1),
serotonin (5-HT) and angiotensin-II (Ang-II).4,10,12–15
Upstream signaling: NE, ET-1, 5-HT and Ang-II
NE. It is generally accepted that penile detumescence and flaccidity are achieved mainly by constant
sympathetic input.2 Both cavernous smooth muscle
(CSM) and the smooth muscle of the penile arteries
and veins are rich in sympathetic innervation. Upon
activation, the sympathetic nerve terminals release
NE, which binds to and stimulates a-adrenoceptors on the smooth muscle membrane.16,17 The
activation of a-adrenoceptors triggers a series of
intracellular signaling pathways involving both
Ca2 þ -dependent12,13 and Ca2 þ -sensitizing mechanisms,10,11 to induce contraction.
ET-1. ET-1, a member of the ET family of peptides,
is among the strongest vasoconstrictors known. ET-1
is produced primarily in the endothelium and plays
an important role in vascular homeostasis.18 In the
corpus cavernosum (CC), ET-1 elicits slow-developing but strong, long-lasting contractions.19 Smooth
muscle cells including CSM cells express two
subtypes of ET-1 receptors: ETA and ETB.19 The
binding of ET-1 to ETA increases vasocontraction,
whereas the binding of ET-1 to ETB leads
to vasorelaxation via the release of NO.19–21 In the
CC, ETA expression is dominant over ETB, and
therefore, ET-1 mainly induces CSM contraction.22,23 Similar to NE, ET-1-induced CSM contraction is mediated by an increase in both [Ca2 þ ]i13,24
and Ca2 þ sensitization.4 Evidence indicates that
changes in the ET-1 pathway are involved in the
pathophysiology of ED. Chang et al.4 found that the
expression of ETA receptors was significantly upregulated in the CSM of diabetic rabbits. Sullivan
et al.25 reported a significant decrease in ETB
receptor binding sites in cavernosal tissue from
hypercholesterolemic rabbits, and a significant increase in ETB receptor binding sites in cavernous
tissue of diabetic rabbits.26
5-HT. 5-HT is a monoamine neurotransmitter that
has a variety of functions in the central nervous
system as well as peripheral tissues. In the CC, 5-HT
released from the serotonergic nerve fibers has a
physiological role in the maintenance of penile
flaccidity and the initiation of detumescence.14,27
Several studies showed that 5-HT induced contraction of isolated penile tissue, which could be
blocked by 5-HT receptor antagonists.14,27–29
Further, pre-incubation with ROCK inhibitor
Y-27632 attenuated maximum contraction induced
by 5-HT of penile tissue, indicating that RhoA/
ROCK pathway is a mediator of 5-HT-induced
contraction.14 Evidence from human pulmonary
International Journal of Impotence Research
arteries showed that Ca2 þ influx and release from
the intracellular Ca2 þ store may also be involved in
5-HT-induced contraction.15
Ang-II. Being an important component of the
renin–Ang system, Ang-II is converted from Ang-I
by Ang-converting enzyme (ACE) predominately in
the lung. Ang-II binds to Ang receptors on the
membranes of smooth muscle cells and other
cell types, causing contraction via both Ca2 þ
-dependent30 and Ca2 þ -sensitizing31 mechanisms,
similar to the other agonists. Becker et al.32,33
reported that Ang-II levels were 30% higher in the
cavernous blood than that in the systemic blood,
indicating that Ang-II is produced locally in the CC.
This is further supported by the findings that ACE
is expressed in the endothelial cells of canine CC.34
The dynamic changes in Ang-II levels in the cavernous blood during different penile states32,33 suggest
that Ang II plays a physiological role in regulating
penile tone. In organ-bath studies, Ang-II evoked dosedependent contraction of human33 and rabbit35 CC
strips. In vivo studies demonstrated that intracavernosal injection (ICI) of Ang-II terminated spontaneous
erections, whereas an Ang II receptor blocker, losartan,
increased the intracavernosal pressure, in a dosedependent manner, in anesthetized dogs.36
Intracellular signaling
Ca2 þ -dependent pathways. Regulation of [Ca2 þ ]i:
It has been widely accepted that elevated [Ca2 þ ]i is
critical for maintaining smooth muscle in a contracted state.37,38 The increase in [Ca2 þ ]i could be a
result of: (1) increased Ca2 þ release from the
intracellular store-sarcoplasmic reticulum (SR);
(2) increased Ca2 þ influx, mainly through the
L-type voltage-gated Ca2 þ channels (VGCCs); and/
or (3) inhibited Ca2 þ removal.39
Ca2 þ release from the SR—There are two types of
Ca2 þ channels in the SR membrane: inositol
trisphosphate (IP3) and ryanodine receptors.40,41
The binding of agonists (NE and others) to their
receptors on the cell membranes activates phospholipase C, which leads to the production of IP3 and
diacylglycerol. IP3 then binds to IP3 receptors on the
SR membrane and triggers the release of Ca2 þ .
These Ca2 þ transients activate Ca2 þ -dependent
Cl channels and depolarize the membrane, and in
turn, open the VGCCs.42,43 The opening of ryanodine receptors is Ca2 þ -dependent, through the
process of Ca2 þ -induced Ca2 þ release, resulting
in a further increase in [Ca2 þ ]i.44 The role of Ca2 þ
release through ryanodine receptors seems to be
more complicated: whereas evidence shows that
they function similarly as the IP3-mediated
Ca2 þ release (that is, mediating contraction),45
other studies indicate that they may activate
Ca2 þ -dependent K þ channels, which in turn causes
Inhibition of smooth muscle tone as a treatment for ED
X Jiang and K Chitaley
hyperpolarization of smooth muscle cells leading
to relaxation.46
Ca2 þ influx through the L-type VGCCs—Although
other types of Ca2 þ channels are involved in Ca2 þ
influx in CSM cells, the L-type VGCCs are believed
to play the leading role in mediating CSM contraction.47 This is supported by the finding that
L-type VGCC blockers relax cavernous tissue strips
contracted with NE.48 The L-type VGCCs open when
the membrane potential increases from the resting
level (90 mV) to 30 mV, resulting from Cl influx
induced by Ca2 þ release from the SR, or Ca2 þ influx
via other types of Ca2 þ channels, including T-type
VGCC.47,49,50 Furthermore, L-type VGCCs can also
be activated when they are phosphorylated by PKC
(discussed below).51,52 The closure of the L-type
VGCC is induced by K þ efflux via Ca2 þ -dependent
K þ channels, and the NO/cGMP pathway.53,54
Ca2 þ removal—Removal of the [Ca2 þ ]i after it has
risen, and maintaining a low background [Ca2 þ ]i, is
mainly achieved by pumping Ca2 þ to the SR lumen
by the sarco(endo)plasmic reticulum Ca2 þ -ATPases,
or to the extracellular spaces by the Na þ –Ca2 þ
exchangers and the plasma membrane Ca2 þ -ATPases.55 It is clear that [Ca2 þ ]i removal can be
promoted by NO/cGMP pathway via activating sarco(endo)plasmic reticulum Ca2 þ -ATPases,56
Na þ –Ca2 þ exchangers57 and plasma membrane
Ca2 þ -ATPases.58 The activity of the sarco(endo)plasmic reticulum Ca2 þ -ATPases in the CC is
significantly higher than that in the bladder and
urethra, and can be significantly downregulated by
castration.59 The role of the Na þ –Ca2 þ exchangers
and the plasma membrane Ca2 þ -ATPases in the
CSM is unclear.
MLCK and MLC: [Ca2 þ ]i binds to calmodulin, and
in turn, the Ca2 þ /calmodulin complex activates
MLCK. MLCK then phosphorylates the regulatory
unit of MLC, allowing it to activate myosin ATPase.
Activity of myosin ATPase permits ratcheting of
myosin and actin myofilaments and muscular contraction. On the contrary, phosphorylated MLC can
be dephosphorylated by MLC phosphatase, resulting in a reversal of contraction.60,61 At basal levels of
tone, only B10% of the MLC in the CC exists in a
phosphorylated state, a significantly lower level than
that in the bladder (25%). Upon maximal stimulation
by phenylephrine, the MLC in the CC reaches a
phosphorylation level of 23%.60 It has been shown
that agonists (NE, ET-1, and so on) also augment
G-protein-dependent downregulation of MLC phosphatase activity, resulting in an increase in the level
of MLC phosphorylation,62 which can be reversed by
activating the NO-cGMP pathway.63,64 The Ca2 þ dependent pathways regulating smooth muscle contraction are summarized in Figure 1.
pathways. RhoA/Rho-kinase:
Ca2 þ -sensitizing
RhoA is a low-molecular-weight G protein, which
Ca
Cl
CDCC
VGCC
R
G protein
51
-
2+
Agonists
PLC
Relaxation
MLC
IP3
DAG
PKC
2+
Ca
CaM
MLCK
MLCP
IP3R
MLC
SR
P
Contraction
Figure 1 Ca2 þ -dependent pathways in smooth muscle cells.
The binding of agonists (NE, ET, and so on) to their receptors on
the cell membrane induces Ca2 þ release from the intracellular
stores and Ca2 þ influx through VGCC. Subsequent increases in
[Ca2 þ ]i result in the activation of myosin light-chain kinase and
phosphorylation of myosin light chain, promoting smooth muscle
contraction. Abbreviations: ET, endothelin; CDCC, Ca2 þ -dependent Cl channels; CaM, calmodulin; DAG, diacylglycerol;
IP3, inositol trisphosphate; IP3R, IP3 receptors; MLC, myosin
light chain; MLCK, myosin light-chain kinase; MLCP, myosin
light-chain phosphatase; NE, norepinephrine; P, phosphate; PKC,
protein kinase C; PLC, phospholipase C; R, receptors; SR,
sarcoplasmic reticulum; VGCC, voltage-gated calcium channels.
is active in the GTP-bound state. The activation of
RhoA requires both its translocation to cellular
membrane and the post-translational addition of a
geranylgeranyl phosphate onto it.65–67 RhoA has
numerous downstream targets, a predominant
one of which is the ser/thr kinase, ROCK. ROCK
has been shown to induce calcium sensitization of
smooth muscle by phosphorylating the myosin
binding subunit of MLC phosphatase, leading to
the inhibition of MLC phosphatase activity.68,69
Some studies also suggest that ROCK may phosphorylate MLC directly.70 ROCK-mediated inhibition of MLC phosphatase leads to the maintenance
of the phosphorylated state of MLC, promoting
vascular smooth muscle contraction. Numerous
studies have demonstrated that the inhibition of
RhoA/ROCK-mediated Ca2 þ sensitization induces
the relaxation of smooth muscle.71–73 In a recent
study, Li et al.74 found that the penile RhoA/ROCK
pathway was upregulated in diabetic rats, and
chronic treatment with the ROCK inhibitor fasudil
could restore erectile function by normalizing the
Akt-driven pathway, indicating that the RhoA/
ROCK pathway plays a pivotal role in the pathogenesis of diabetic ED.
There are two isoforms of ROCK: ROCK1 (ROKb)
and ROCK2 (ROKa). In humans, ROCK1 and ROCK2
genes are located separately on chromosomes 18
and 2, respectively.75,76 ROCK1 is preferentially
expressed in inflammatory cells, whereas ROCK2
is highly expressed in vascular smooth muscle
cells.77 Wang et al.78 reported that whereas a balance
of ROCK1 and ROCK2 activities is required to
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Inhibition of smooth muscle tone as a treatment for ED
X Jiang and K Chitaley
52
regulate vascular smooth muscle actin cytoskeletal
structure, ROCK2 is the predominant isoform that
regulates vascular smooth muscle cell contractility.
Both ROCK1 and ROCK2 are expressed in human
and animal CC.79,80 Elevated expression of the
ROCK2 protein was found in the cavernous tissue
of spontaneous hypertensive rats81 and rats that had
undergone cavernous nerve injury,82 in line with the
findings that ROCK2 plays the major role in
regulating vascular smooth muscle cell contractility.
However, several studies indicate that the expression of ROCK1, rather than ROCK2, was significantly increased in penile tissues from different
diabetic animal models.4,83,84 In rabbits with bladder outlet obstruction, Chang et al.80 found that the
expression of both isoforms of ROCK in the CC is
increased. The etiology-dependent changes in the
expression of ROCK1 and ROCK2 indicate that they
might play different roles in the pathophysiology of
ED, which may have implications in the development of therapeutic options.
Telokin: Telokin, also known as kinase-related
protein, is a 17-kDa smooth muscle-specific protein
whose sequence is identical to the C-terminal
domain of MLCK.85,86 It has been shown that telokin
decreases smooth muscle contractility by inhibiting
the phosphorylation of the regulatory unit of MLC by
the MLCK.87–90 Increasing evidence shows that
telokin also activates MLC phosphatase, and therefore, leads to Ca2 þ desensitization.85,91–94 Telokin
knockout mice exhibit decreased MLC phosphatase
activity, resulting in increased Ca2 þ sensitivity in
intestinal smooth muscle.94 The activity and function
of telokin in the cavernous tissue remains unclear.
PKC: PKC is a family of enzymes that are involved
in controlling the function of other proteins through
the phosphorylation of hydroxyl groups of serine
and threonine amino-acid residues on these proteins. Activated by signals such as diacylglycerol or
Ca2 þ , PKC regulates smooth muscle tone via
complex and diverse signal-transduction cascades.
First, PKC regulates the activity of L-type VGCCs,
non-selective cation transient receptor potential
channels, Ca2 þ -activated K þ channels and ATPsensitive K þ channels by phosphorylating them.95
The function of PKC could be either activating or
inhibiting these ion channels, depending on the cell
type, the PKC isoforms involved and the concentration of PKC agonist.95–97 Another mechanism by
which PKC regulates smooth muscle tone is to
increase Ca2 þ sensitization via phosphorylation of
CPI-17 (discussed next). In addition, PKC was found
to inhibit NO synthase activity.98–100 Increased
PKC activity in diabetic human cavernous tissue
has been reported,101 indicating that the alteration of
PKC activity might be involved in the pathogenesis
of ED. However, Jin et al.102 failed to detect any
significant effect of a PKC activator or PKC inhibitors on the tone of mouse cavernous tissue, although
there were significant effects on mouse aorta.
International Journal of Impotence Research
Agonists
PLC
R
G protein
RhoA-GTP
CPI-17
IP3 DAG
PKC
ROCK
CPI-17
P
MLCP
Contraction
MLC Relaxation
MLC
P
MLCK
Figure 2 Ca2 þ -sensitizing pathways in smooth muscle cells.
The binding of agonists (NE, ET, and so on) to their receptors on
the cell membrane induces activation of RhoA. RhoA-GTP in turn
activates ROCK, leading to the inhibition of MLC phosphatase
activity by phosphorylation of the myosin binding subunit of
MLC phosphatase. Furthermore, both PKC and ROCK can
phosphorylate and activate CPI-17, which also inhibits MLC
phosphatase activity, to promote smooth muscle contraction.
Abbreviations: ET, endothelin; DAG, diacylglycerol; IP3, inositol
trisphosphate; MLC, myosin light chain; MLCP, myosin lightchain phosphatase; MLCK, myosin light-chain kinase; NE,
norepinephrine; P, phosphate; PLC, phospholipase C; PKC,
protein kinase C; R, receptors; ROCK, Rho-kinase.
CPI-17: CPI-17, known as PKC potentiated inhibitory protein of protein phosphatase-1, is a 17-kDa
protein that can be phosphorylated primarily by
PKC, although other kinases, such as ROCK and
p21-activated protein kinase, have been suggested to
phosphorylate CPI-17 as well.103–106 Phosphorylation at Thr-38 greatly increases the inhibitory
potency of CPI-17, which in turn inhibits MLC
phosphatase activity,107 leading to increased phosphorylation of MLC and contraction of smooth
muscle.108 The phosphorylation of CPI-17 was
diminished in decompensated bladder tissue, indicating that the activity level of CPI-17 is correlated
with smooth muscle contractility.109 CPI-17 expression was detected in human and rabbit CC;79
however, its role in penile erection remains to be
determined. Figure 2 illustrates the Ca2 þ -sensitizing
pathways regulating smooth muscle contraction.
Contractile signaling and ED
Diabetic ED
Several lines of evidence showed that diabetic ED is
associated with an imbalance towards enhanced
penile vasoconstriction, resulting from changes
in contractile signaling of the CSM at multiple
levels. The concentration of penile NE was found
to be increased in diabetic rats,110 and elevated
plasma level of ET-1 has been shown in diabetic
patients.111,112 Chang et al.5 found that the expression of ETA receptors was significantly upregulated
Inhibition of smooth muscle tone as a treatment for ED
X Jiang and K Chitaley
in the CC of diabetic rabbits and rats.84 Sullivan
et al.26 reported a significant increase in ETB
receptor binding sites in cavernous tissue of diabetic
rabbits. As the downstream signaling of both NE and
ET, RhoA/ROCK axis was found to be upregulated in
diabetic animals as well. The expression of ROCK1,
but not ROCK2, was significantly increased in
penile tissues from different diabetic animal models,4,83,84 indicating that ROCK1 is involved in
diabetic ED. Furthermore, Angulo et al.101 demonstrated that diabetes causes hypercontractility of
human cavernous tissue by a mechanism involving
overactivity of PKC.
Hypertension-related ED
Similar to diabetic ED, hypertension-related ED may
be related to augmented contractile signaling in the
CC as well. Deoxycorticosterone acetate-salt-induced hypertensive animals showed increased
cavernous contractile responses to both ET-1 and
the a-adrenergic receptor agonist phenylephrine.113,114 As to the downstream signaling, although
protein expression levels of ROCK in the CC of
deoxycorticosterone acetate-treated animals were
similar to those of control animals, the phosphorylated form of myosin phosphatase regulatory subunit, a downstream effector of ROCK, was increased
in cavernous tissue from deoxycorticosterone acetate animals.114 In the spontaneously hypertensive
rat, Wilkes et al.115 found that hypertension-related
ED is associated with elevated penile RhoA levels
and that inhibition of ROCK activity with Y-27632
was beneficial in attenuating the decline in erectile
function. In another study, ROCK2 protein was
found to be elevated in spontaneous hypertensive
rats.81 However, Behr-Roussel et al.116 found both a
reduced cavernous contractile response to phenylepherine and an impaired endothelium-dependent
relaxation to acetylcholine in spontaneous hypertensive rats. The authors proposed that the pathophysiology of ED in hypertension is the result of an
increase in cyclooxygenase-dependent constrictor
tone, although a defect of endothelial or neuronal
NO production and/or bioavailability cannot be
excluded.
Aging-related ED
Aging is closely related to ED, and interestingly,
despite an increased contractile response in the
CC,117 aged animals showed decreased (rather than
increased) penile concentration of NE110 and
expression of L-type VGCC and ryanodine receptors.118 It is possible that aging mainly impairs the
relaxation signaling, resulting in ‘unmasking’ of the
contractile signaling. However, Jin et al.119 reported
that though the expression of total RhoA remains
unchanged, membrane-bound RhoA and phos-
phorylated myosin phosphatase regulatory subunit
in the CC of aged rats are increased, indicating that
enhanced RhoA and ROCK activity may play a
role. Treatment with an Ang-II receptor antagonist120
and the ROCK inhibitor Y-27632121 also improve
erectile function in aged animals, indicating
that elevated contractile tone may be involved in
aging-related ED.
53
ED caused by cavernous nerve injury
Cavernous nerve injury is another common cause of
ED. Following cavernous nerve injury, animals show
penile fibrosis, that is, decreased smooth muscle
and increased collagen content.122 Gratzke et al.82
reported that bilateral cavernous nerve injury causes
increased RhoA and ROCK2 protein expression, and
increased RhoA GTPase and ROCK activity in rat
CC. The penile ROCK1 protein expression is unchanged in these animals. However, Cho et al.123
found increased ROCK1 expression in the CC of rats
following cavernous nerve injury. In accordance
with these findings, ICI of Y-27632 causes a
significantly greater increase in intracavernosal
pressure in nerve-injured rats compared to that
in sham-operated rats.123
Inhibition of vasoconstriction to induce
erection
Endogenous mechanisms
The main driving force for penile erection is
NO-mediated signaling. Sexual stimulation or
nocturnal tumescence activates neuronal NO
synthase-mediated NO release from non-adrenergic
non-cholinergic nerve endings8,124 initiating vasodilation. Subsequently, maintenance of dilation
has been proposed to occur through sheer flowinduced endothelial NOS activation.125 In the
smooth muscle cell, NO stimulates soluble guanylate cyclase, activating cGMP-dependent protein
kinase for cavernosal relaxation.126
NO signaling was originally thought to induce
vasodilation by modulation of [Ca2 þ ]i, through
events including the inhibition of L-type VGCCs,
the activation of Ca2 þ -dependent K þ channels, the
promotion of plasma membrane Ca2 þ -ATPases and
Na þ –Ca2 þ exchangers activity and the activation
of
sarco(endo)plasmic
reticulum
Ca2 þ -ATP
56,127,128
It is now clear that NO signaling can
ases.
also inhibit Ca2 þ -sensitizing mechanisms directly
resulting in vasorelaxation. cGMP-dependent protein kinase can decrease levels of phosphorylated
MLC through telokin-mediated activation of MLC
phosphatase,93 as well as direct inhibition of RhoA
through its phosphorylation.129,130 Recombinant
cGMP-dependent protein kinase has been shown
to phosphorylate RhoA, destabilizing its membrane
International Journal of Impotence Research
Inhibition of smooth muscle tone as a treatment for ED
X Jiang and K Chitaley
54
binding resulting in NO-mediated inhibit of RhoA/
ROCK activity.129,130 It is tempting to speculate that
decreased NO bioavailability leads to an increase in
RhoA/ROCK constrictor activity. Elevated ROCK
activity may then mediate the increased vasoconstrictor sensitivity seen in various animal models of
ED. Endogenously, decreased [Ca2 þ ]i induced by
NO may also lead to vasodilation through inactivation of PKC, rapid CPI-17 dephosphorylation as well
as MLCK inactivation, resulting in rapid MLC
dephosphorylation and relaxation.131
Pharmacological therapies
Blockage of upstream signaling (NE, ET-1, ANG-II).
NE: The non-selective a-adrenoceptor blocker,
phentolamine, can block the effect of NE by
competitively binding to the a1-adrenoceptor in
the CC, leading to the relaxation of the CSM.132 In
addition to the blockage of a-adrenoceptors, phentolamine may also induce relaxation of cavernous
tissue by blocking ET-1 signaling.133 However, ICI
with 5 mg phentolamine only resulted in penile
tumescence, but not rigidity in humans.5 Currently,
phentolamine is administered in conjunction with
other vasoactive agents, such as papaverine, prostaglandin E1 and vasoactive intestinal polypeptide5
as ICI therapies, with the advantages of lowering
the dosages and therefore the adverse effects of the
other agents.134 The safety and efficacy of oral
phentolamine in the treatment of ED have been
explored.135,136 Compared to sildenafil, oral phentolamine has a higher incidence of adverse effects
and is less effective in improving penile erection.136
Selective a1-adrenoceptor antagonists are commonly used to treat lower urinary tract symptoms.
Studies showed that they slightly improved erectile function as well.137,138 An additive effect was
observed when the selective a1-adrenoceptor antagonist alfuzosin was used together with sildenafil to
treat ED.139
ET-1: An ETA receptor antagonist was shown to
increase the duration of nerve-stimulated elevations
in intracavernosal pressure in rabbits, although the
peak pressure values were not altered.6 Despite the
efficacy of the ETA receptor antagonist in animal
studies, oral administration of this drug to men with
mild-to-moderate ED did not significantly improve
erectile function compared to placebo.6 This disparity in efficacy between lab studies in rabbits and
clinical studies was possibly due to important
differences between species with regard to the role
of ET-1 in erectile function. This is further supported by the findings in rats that ET receptor
antagonists do not significantly alter the erectile
response, although they inhibit potent contractions
to exogenous ET-1.140 Nevertheless, treatment with
ET receptor antagonists for 2 weeks reversed
cavernous apoptosis in diabetic rats,141 indicating
International Journal of Impotence Research
that chronic administration of ET receptor antagonists might be beneficial in the protection of erectile
tissue.
Ang-II: ACE inhibitors and Ang-II receptor blockers are widely used antihypertensive drugs. Since
blockage of the Ang-II pathway results in smooth
muscle relaxation, these agents might be beneficial
in the treatment of ED. Animal studies showed that
Ang-II receptor antagonists improved penile function in aged animals or animals with dyslipidemia.120,142 Another study showed that the ACE
inhibitor captopril corrected both the blood pressure
and erectile response of hypertensive rats to control
levels.143 Several small clinical studies have also
suggested that treatment with Ang-II receptor blockers or ACE inhibitors are associated with improved
erectile function and sexual performance in patients
with hypertension and diabetes mellitus.144–146
However, a recent double-blind, randomized study,
involving 1549 patients, failed to reveal any significant effect of an Ang-II receptor blockers or an
ACE inhibitor on ED.147
Blockage of the accumulation of [Ca2 þ ]i. Given
that [Ca2 þ ]i plays a central role in mediating CSM
contraction, drugs that inhibit the increase in
[Ca2 þ ]i may be potentially effective in the treatment
of ED. This is supported by the findings that calcium
channel blockers (CCBs) significantly relaxed rabbit48 and human148,149 CSM contracted by a-adrenergic agonists in vitro. However, ICI of CCBs in
dogs was less effective and had more side effects
compared to papaverine.150 The results of clinical
trials with oral CCBs in the treatment of ED are
disappointing. CCBs exert either no effect or a
negative effect on erectile function.151,152 The safety
and efficacy of ICI of CCBs have also been
studied.153,154 Although the side effect of ICI of
CCBs was comparable to other agents, CCBs may not
be as effective as blockade of a-adrenoceptors.148,153,154
Blockage of Ca2 þ -sensitizing pathway (mainly
RhoA/ROCK). Ten years ago, the inhibition of
ROCK by ICI of Y-27632 was found to induce an
erectile response in rats.7 This effect did not require
activation of NO-mediated signaling, thus introducing a potential target pathway for the treatment of
ED, with potential extended benefit in cases where
NO bioavailability was attenuated and thus phosphodiesterase type 5 (PDE5) inhibitors were less
efficacious. At this point, ROCK inhibitors are being
developed and tested for a wide range of potential
uses. In Japan, the ROCK inhibitor fasudil has been
used to treat vasospasm following subarachnoid
hemorrhage155 and pulmonary hypertension.156 One
animal study showed that chronic administration of
oral fasudil prevented the development of ED
and pelvic atherosclerosis.157 Another study using
Inhibition of smooth muscle tone as a treatment for ED
X Jiang and K Chitaley
55
Table 1 Summary of study outcomes on targets for pharmacological intervention
Category
a-Adrenoceptor antagonists
Animal studies
Human studies
ICI
Systemic
ICI
Oral
m ICP
—
Tumescence; used in
adjunction with
other agents
Mildly improve
erection
ET receptor antagonists
m the duration of erection
k apoptosis in diabetic CC
—
Non-efficacious
ACE inhibitors
—
Correct hypertensive ED
—
Non-efficacious
Ang-II receptor blockers
m ICP
m erection in aged and dyslipidemia
animals
—
Non-efficacious
Calcium channel blockers
Less effective than
papaverine
—
Non-efficacious
Non-efficacious
ROCK inhibitors
Induce erectile response
Prevent ED; restore erectile function
—
—
Abbreviations: ACE, angiotensin-converting enzyme; Ang, angiotensin; ED, erectile dysfunction; ET, endothelin; ICI, intracavernosal
injection; ICP, intracavernosal pressure; ROCK, Rho-kinase.
diabetic rat models demonstrated that fasudil restores erectile function by suppressing corporal
apoptosis caused by diabetes.74 These promising
results from animal studies indicate the possibilities
of administration of ROCK inhibitors either acutely
to induce erection or chronically to prevent or
reverse ED. To date, the effect of ROCK inhibitors
in the treatment of ED in humans has not been
reported. In the era of oral therapy for ED, ICI
therapy becomes less attractive. However, one
potential problem with systemic administration of
ROCK inhibitors is whether it would cause profound extra-cavernosous effects, such as hypotension. Further, the two ROCK isoforms share B90%
homology in their kinase domains.158 This has made
it quite challenging to develop isoform-selective
inhibitors for clinical use. A recent compound,
SLx2119 (Surface Logix), is unique in its specificity
for only ROCK2.159
A summary of the outcomes of both animal and
human studies on the pharmacological targets modulating CSM contraction for the treatment of ED is
shown in Table 1. The most significant advances
during the past decade are listed as follows:
a-Adrenoceptor antagonists have an additive
effect when administered together with other ED
medications.
Chronic administration of ET receptor antagonists
is beneficial in the protection of erectile tissue in
rats. However, data in humans are lacking.
Chronic administration of ROCK inhibitors prevents ED or restores erectile function in animals.
However, clinical studies are needed.
the need for intact NO-dependent signaling, is
especially appealing for the treatment of ED associated with co-morbidities such as diabetes or
prostatectomy, where NO signaling may be impaired. However, more than 10 years after the
discovery of potent contractile signaling pathways
in the cavernosum, we remain far from such targeted
therapeutics. The most promise for therapeutic
intervention has been found with the inhibitor of
ROCK signaling. The availability of the ROCK
inhibitor fasudil in Japan for the treatment of other
conditions makes clinical studies of its efficacy and
safety in the treatment of ED possible. Furthermore,
isoform-selective ROCK inhibitors may be indicated
in the treatment of ED with different etiologies with
a reduced systemic side effect, although various
obstacles, such as isoform homology and lack of
tissue specificity, have hindered their development.
Other potential targets in both Ca2 þ -dependent
and Ca2 þ -sensitizing signaling pathways may be
explored for benefits in the treatment of ED.
Combined blockages of multiple targets in these
pathways may enhance the efficacy in promoting
penile erection. Development of these targeted
therapeutics, which may benefit even cohorts
refractory to PDE5 inhibition, are certainly promising and warranted.
Conflict of interest
The authors declare no conflict of interest.
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Conclusions
This review outlines contractile signaling pathways,
which play a role in the maintenance of penile
flaccidity. The potential for therapeutic intervention
at the level of smooth muscle contraction, bypassing
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