The Promise of Inhibition of Smooth Muscle Tone As a Treatment for Erectile Dysfunction: Where Are We Now?

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 (NO)-mediated pathways), may be a beneficial strategy for the treatment of ED.5–7 Erectile dysfunction (ED) affects 30 million men In this review, we will outline the major smooth in the United States1 and is associated with muscle signaling pathways involved in penile co-morbidities ranging from prostatectomy to dia- vasoconstriction and discuss the potential for betes and increased age. Penile erection is a inhibition of these pathways as a treatment option dynamic process requiring dilation of feeder arter- for organic ED. A focus will be on the promise ioles and cavernosal sinusoids allowing for in- and limitations of pharmacological therapy based on creased inflow of blood. It is important to current progress in the development of RhoA/ remember, however, that the vast majority of the Rho-kinase (ROCK) antagonists. time, these arterioles and sinusoids are maintained in the collapsed/contracted state, severely restrict- ing penile blood flow.2 The maintenance of penile Contractile signaling flaccidity through vasomotor tone is an active process involving complex signaling mechanisms. The vast majority of time, the penis is maintained in Heightened smooth muscle contraction is present in the flaccid state through active contraction of penile 3,4 some models of ED, and various studies have arterioles. The release of norepinephrine (NE) from suggested that pharmacological inhibition of sympathetic nerve terminals activates arteriolar and smooth muscle contraction, as opposed to active cavernosal a-adrenergic receptors.2,8,9 Subsequent 2 þ 2 þ induction of dilation (such as through nitric oxide increases in intracellular Ca concentration ([Ca ]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 Correspondence: Dr K Chitaley, Department of Urology, 2 þ University of Washington, Box 358050, 815 Mercer Street, mechanism, Ca -sensitizing pathways, such as Room 319, Seattle, WA 98109, USA. ROCK- and protein kinase C (PKC)-mediated signal- E-mail: [email protected] ing, can promote contraction through the inhibition Received 28 March 2011; revised 13 July 2011; accepted of MLC phosphatase or the direct stimulation of 17 August 2011; published online 6 October 2011 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 arteries showed that Ca2 þ influx and release from NE or other agonists, including endothelin-1 (ET-1), the intracellular Ca2 þ store may also be involved in serotonin (5-HT) and angiotensin-II (Ang-II).4,10,12–15 5-HT-induced contraction.15

Ang-II. Being an important component of the Upstream signaling: NE, ET-1, 5-HT and Ang-II renin–Ang system, Ang-II is converted from Ang-I NE. It is generally accepted that penile detumes- by Ang-converting enzyme (ACE) predominately in cence and flaccidity are achieved mainly by constant the lung. Ang-II binds to Ang receptors on the sympathetic input.2 Both cavernous smooth muscle membranes of smooth muscle cells and other (CSM) and the smooth muscle of the penile arteries cell types, causing contraction via both Ca2 þ and veins are rich in sympathetic innervation. Upon -dependent30 and Ca2 þ -sensitizing31 mechanisms, activation, the sympathetic nerve terminals release similar to the other agonists. Becker et al.32,33 NE, which binds to and stimulates a-adreno- reported that Ang-II levels were 30% higher in the ceptors on the smooth muscle membrane.16,17 The cavernous blood than that in the systemic blood, activation of a-adrenoceptors triggers a series of indicating that Ang-II is produced locally in the CC. intracellular signaling pathways involving both This is further supported by the findings that ACE Ca2 þ -dependent12,13 and Ca2 þ -sensitizing mecha- is expressed in the endothelial cells of canine CC.34 nisms,10,11 to induce contraction. The dynamic changes in Ang-II levels in the cavernous blood during different penile states32,33 suggest ET-1. ET-1, a member of the ET family of peptides, that Ang II plays a physiological role in regulating penile tone. In organ-bath studies, Ang-II evoked dose- is among the strongest vasoconstrictors known. ET-1 33 35 is produced primarily in the endothelium and plays dependent contraction of human and rabbit CC an important role in vascular homeostasis.18 In the strips. In vivo studies demonstrated that intracaverno- corpus cavernosum (CC), ET-1 elicits slow-develop- sal injection (ICI) of Ang-II terminated spontaneous ing but strong, long-lasting contractions.19 Smooth erections, whereas an Ang II receptor blocker, losartan, increased the intracavernosal pressure, in a dose- muscle cells including CSM cells express two 36 19 dependent manner, in anesthetized dogs. subtypes of ET-1 receptors: ETA and ETB. The binding of ET-1 to ETA increases vasocontraction, whereas the binding of ET-1 to ETB leads 19–21 to vasorelaxation via the release of NO. In the Intracellular signaling CC, ET expression is dominant over ET , and 2 þ 2 þ A B Ca -dependent pathways. Regulation of [Ca ]i: therefore, ET-1 mainly induces CSM contrac- 2 þ It has been widely accepted that elevated [Ca ]i is tion.22,23 Similar to NE, ET-1-induced CSM contrac- 2 þ 13,24 critical for maintaining smooth muscle in a con- tion is mediated by an increase in both [Ca ]i 37,38 2 þ 2 þ 4 tracted state. The increase in [Ca ]i could be a and Ca sensitization. Evidence indicates that result of: (1) increased Ca2 þ release from the changes in the ET-1 pathway are involved in the 4 intracellular store-sarcoplasmic reticulum (SR); pathophysiology of ED. Chang et al. found that the (2) increased Ca2 þ influx, mainly through the expression of ETA receptors was significantly upre- L-type voltage-gated Ca2 þ channels (VGCCs); and/ gulated in the CSM of diabetic rabbits. Sullivan 2 þ 39 25 or (3) inhibited Ca removal. et al. reported a significant decrease in ETB Ca2 þ release from the SR—There are two types of receptor binding sites in cavernosal tissue from Ca2 þ channels in the SR membrane: inositol hypercholesterolemic rabbits, and a significant in- 40,41 trisphosphate (IP3) and ryanodine receptors. crease in ETB receptor binding sites in cavernous 26 The binding of agonists (NE and others) to their tissue of diabetic rabbits. receptors on the cell membranes activates phospholipase C, which leads to the production of IP3 and 5-HT. 5-HT is a monoamine neurotransmitter that diacylglycerol. IP3 then binds to IP3 receptors on the has a variety of functions in the central nervous SR membrane and triggers the release of Ca2 þ . system as well as peripheral tissues. In the CC, 5-HT These Ca2 þ transients activate Ca2 þ -dependent released from the serotonergic nerve fibers has a ClÀ channels and depolarize the membrane, and in physiological role in the maintenance of penile turn, open the VGCCs.42,43 The opening of ryano- flaccidity and the initiation of detumescence.14,27 dine receptors is Ca2 þ -dependent, through the Several studies showed that 5-HT induced contrac- process of Ca2 þ -induced Ca2 þ release, resulting 2 þ 44 2 þ tion of isolated penile tissue, which could be in a further increase in [Ca ]i. The role of Ca blocked by 5-HT receptor antagonists.14,27–29 release through ryanodine receptors seems to be Further, pre-incubation with ROCK inhibitor more complicated: whereas evidence shows that Y-27632 attenuated maximum contraction induced they function similarly as the IP3-mediated by 5-HT of penile tissue, indicating that RhoA/ Ca2 þ release (that is, mediating contraction),45 ROCK pathway is a mediator of 5-HT-induced other studies indicate that they may activate contraction.14 Evidence from human pulmonary Ca2 þ -dependent K þ channels, which in turn causes

International Journal of Impotence Research Inhibition of smooth muscle tone as a treatment for ED X Jiang and K Chitaley 51 2+ - hyperpolarization of smooth muscle cells leading Agonists Ca Cl 46 to relaxation. VGCC CDCC 2 þ R Ca influx through the L-type VGCCs—Although G protein PLC 2 þ 2 þ other types of Ca channels are involved in Ca Relaxation influx in CSM cells, the L-type VGCCs are believed MLC to play the leading role in mediating CSM contr- IP DAG PKC 47 3 action. This is supported by the finding that 2+ L-type VGCC blockers relax cavernous tissue strips Ca CaM MLCK MLCP contracted with NE.48 The L-type VGCCs open when IP R the membrane potential increases from the resting 3 À level (À90 mV) to À30 mV, resulting from Cl influx MLC 2 þ 2 þ induced by Ca release from the SR, or Ca influx SR P via other types of Ca2 þ channels, including T-type Contraction VGCC.47,49,50 Furthermore, L-type VGCCs can also be activated when they are phosphorylated by PKC Figure 1 Ca2 þ -dependent pathways in smooth muscle cells. (discussed below).51,52 The closure of the L-type The binding of agonists (NE, ET, and so on) to their receptors on þ 2 þ the cell membrane induces Ca2 þ release from the intracellular VGCC is induced by K efflux via Ca -dependent 2 þ þ 53,54 stores and Ca influx through VGCC. Subsequent increases in K channels, and the NO/cGMP pathway. 2 þ 2 þ 2 þ [Ca ]i result in the activation of myosin light-chain kinase and Ca removal—Removal of the [Ca ]i after it has phosphorylation of myosin light chain, promoting smooth muscle 2 þ 2 þ risen, and maintaining a low background [Ca ]i,is contraction. Abbreviations: ET, endothelin; CDCC, Ca -depen- mainly achieved by pumping Ca2 þ to the SR lumen dent ClÀ channels; CaM, calmodulin; DAG, diacylglycerol; by the sarco(endo)plasmic reticulum Ca2 þ -ATPases, IP3, inositol trisphosphate; IP3R, IP3 receptors; MLC, myosin þ 2 þ light chain; MLCK, myosin light-chain kinase; MLCP, myosin or to the extracellular spaces by the Na –Ca light-chain phosphatase; NE, norepinephrine; P, phosphate; PKC, 2 þ exchangers and the plasma membrane Ca -ATP- protein kinase C; PLC, phospholipase C; R, receptors; SR, 55 2 þ ases. It is clear that [Ca ]i removal can be sarcoplasmic reticulum; VGCC, voltage-gated calcium channels. promoted by NO/cGMP pathway via activating sarco(endo)plasmic reticulum Ca2 þ -ATPases,56 Na þ –Ca2 þ exchangers57 and plasma membrane is active in the GTP-bound state. The activation of Ca2 þ -ATPases.58 The activity of the sarco(endo)- RhoA requires both its translocation to cellular plasmic reticulum Ca2 þ -ATPases in the CC is membrane and the post-translational addition of a significantly higher than that in the bladder and geranylgeranyl phosphate onto it.65–67 RhoA has urethra, and can be significantly downregulated by numerous downstream targets, a predominant castration.59 The role of the Na þ –Ca2 þ exchangers one of which is the ser/thr kinase, ROCK. ROCK and the plasma membrane Ca2 þ -ATPases in the has been shown to induce calcium sensitization of CSM is unclear. smooth muscle by phosphorylating the myosin 2 þ MLCK and MLC:[Ca ]i binds to calmodulin, and binding subunit of MLC phosphatase, leading to in turn, the Ca2 þ /calmodulin complex activates the inhibition of MLC phosphatase activity.68,69 MLCK. MLCK then phosphorylates the regulatory Some studies also suggest that ROCK may phos- unit of MLC, allowing it to activate myosin ATPase. phorylate MLC directly.70 ROCK-mediated inhibi- Activity of myosin ATPase permits ratcheting of tion of MLC phosphatase leads to the maintenance myosin and actin myofilaments and muscular con- of the phosphorylated state of MLC, promoting traction. On the contrary, phosphorylated MLC can vascular smooth muscle contraction. Numerous be dephosphorylated by MLC phosphatase, resul- studies have demonstrated that the inhibition of ting in a reversal of contraction.60,61 At basal levels of RhoA/ROCK-mediated Ca2 þ sensitization induces tone, only B10% of the MLC in the CC exists in a the relaxation of smooth muscle.71–73 In a recent phosphorylated state, a significantly lower level than study, Li et al.74 found that the penile RhoA/ROCK that in the bladder (25%). Upon maximal stimulation pathway was upregulated in diabetic rats, and by phenylephrine, the MLC in the CC reaches a chronic treatment with the ROCK inhibitor fasudil phosphorylation level of 23%.60 It has been shown could restore erectile function by normalizing the that agonists (NE, ET-1, and so on) also augment Akt-driven pathway, indicating that the RhoA/ G-protein-dependent downregulation of MLC phos- ROCK pathway plays a pivotal role in the pathogen- phatase activity, resulting in an increase in the level esis of diabetic ED. of MLC phosphorylation,62 which can be reversed by There are two isoforms of ROCK: ROCK1 (ROKb) activating the NO-cGMP pathway.63,64 The Ca2 þ - and ROCK2 (ROKa). In humans, ROCK1 and ROCK2 dependent pathways regulating smooth muscle con- genes are located separately on chromosomes 18 traction are summarized in Figure 1. and 2, respectively.75,76 ROCK1 is preferentially expressed in inflammatory cells, whereas ROCK2 is highly expressed in vascular smooth muscle Ca2 þ -sensitizing pathways. RhoA/Rho-kinase: cells.77 Wang et al.78 reported that whereas a balance RhoA is a low-molecular-weight G protein, which of ROCK1 and ROCK2 activities is required to

International Journal of Impotence Research Inhibition of smooth muscle tone as a treatment for ED X Jiang and K Chitaley 52 regulate vascular smooth muscle actin cytoskeletal Agonists structure, ROCK2 is the predominant isoform that R regulates vascular smooth muscle cell contractility. PLC G protein RhoA-GTP 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 CPI-17 81 IP DAG PKC ROCK of spontaneous hypertensive rats and rats that had 3 82 CPI-17 undergone cavernous nerve injury, in line with the P findings that ROCK2 plays the major role in

regulating vascular smooth muscle cell contractility. MLCP However, several studies indicate that the expression of ROCK1, rather than ROCK2, was signifi- Contraction MLC MLC Relaxation P cantly increased in penile tissues from different MLCK diabetic animal models.4,83,84 In rabbits with blad- 2 þ der outlet obstruction, Chang et al.80 found that the Figure 2 Ca -sensitizing pathways in smooth muscle cells. The binding of agonists (NE, ET, and so on) to their receptors on expression of both isoforms of ROCK in the CC is the cell membrane induces activation of RhoA. RhoA-GTP in turn increased. The etiology-dependent changes in the activates ROCK, leading to the inhibition of MLC phosphatase expression of ROCK1 and ROCK2 indicate that they activity by phosphorylation of the myosin binding subunit of might play different roles in the pathophysiology of MLC phosphatase. Furthermore, both PKC and ROCK can ED, which may have implications in the develop- phosphorylate and activate CPI-17, which also inhibits MLC phosphatase activity, to promote smooth muscle contraction. ment of therapeutic options. Abbreviations: ET, endothelin; DAG, diacylglycerol; IP3, inositol Telokin: Telokin, also known as kinase-related trisphosphate; MLC, myosin light chain; MLCP, myosin light- protein, is a 17-kDa smooth muscle-specific protein chain phosphatase; MLCK, myosin light-chain kinase; NE, whose sequence is identical to the C-terminal norepinephrine; P, phosphate; PLC, phospholipase C; PKC, protein kinase C; R, receptors; ROCK, Rho-kinase. 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 CPI-17: CPI-17, known as PKC potentiated inhibi- telokin also activates MLC phosphatase, and there- tory protein of protein phosphatase-1, is a 17-kDa fore, leads to Ca2 þ desensitization.85,91–94 Telokin protein that can be phosphorylated primarily by knockout mice exhibit decreased MLC phosphatase PKC, although other kinases, such as ROCK and activity, resulting in increased Ca2 þ sensitivity in p21-activated protein kinase, have been suggested to intestinal smooth muscle.94 The activity and function phosphorylate CPI-17 as well.103–106 Phosphoryla- of telokin in the cavernous tissue remains unclear. tion at Thr-38 greatly increases the inhibitory PKC: PKC is a family of enzymes that are involved potency of CPI-17, which in turn inhibits MLC in controlling the function of other proteins through phosphatase activity,107 leading to increased phos- the phosphorylation of hydroxyl groups of serine phorylation of MLC and contraction of smooth and threonine amino-acid residues on these pro- muscle.108 The phosphorylation of CPI-17 was teins. Activated by signals such as diacylglycerol or diminished in decompensated bladder tissue, indi- Ca2 þ , PKC regulates smooth muscle tone via cating that the activity level of CPI-17 is correlated complex and diverse signal-transduction cascades. with smooth muscle contractility.109 CPI-17 expres- First, PKC regulates the activity of L-type VGCCs, sion was detected in human and rabbit CC;79 non-selective cation transient receptor potential however, its role in penile erection remains to be channels, Ca2 þ -activated K þ channels and ATP- determined. Figure 2 illustrates the Ca2 þ -sensitizing sensitive K þ channels by phosphorylating them.95 pathways regulating smooth muscle contraction. 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 Contractile signaling and ED which PKC regulates smooth muscle tone is to increase Ca2 þ sensitization via phosphorylation of Diabetic ED CPI-17 (discussed next). In addition, PKC was found Several lines of evidence showed that diabetic ED is to inhibit NO synthase activity.98–100 Increased associated with an imbalance towards enhanced PKC activity in diabetic human cavernous tissue penile vasoconstriction, resulting from changes has been reported,101 indicating that the alteration of in contractile signaling of the CSM at multiple PKC activity might be involved in the pathogenesis levels. The concentration of penile NE was found of ED. However, Jin et al.102 failed to detect any to be increased in diabetic rats,110 and elevated significant effect of a PKC activator or PKC inhibi- plasma level of ET-1 has been shown in diabetic tors on the tone of mouse cavernous tissue, although patients.111,112 Chang et al.5 found that the expres- there were significant effects on mouse aorta. sion of ETA receptors was significantly upregulated

International Journal of Impotence Research Inhibition of smooth muscle tone as a treatment for ED X Jiang and K Chitaley 53 in the CC of diabetic rabbits and rats.84 Sullivan phorylated myosin phosphatase regulatory subunit 26 et al. reported a significant increase in ETB in the CC of aged rats are increased, indicating that receptor binding sites in cavernous tissue of diabetic enhanced RhoA and ROCK activity may play a rabbits. As the downstream signaling of both NE and role. Treatment with an Ang-II receptor antagonist120 ET, RhoA/ROCK axis was found to be upregulated in and the ROCK inhibitor Y-27632121 also improve diabetic animals as well. The expression of ROCK1, erectile function in aged animals, indicating but not ROCK2, was significantly increased in that elevated contractile tone may be involved in penile tissues from different diabetic animal mod- aging-related ED. els,4,83,84 indicating that ROCK1 is involved in diabetic ED. Furthermore, Angulo et al.101 demonstrated that diabetes causes hypercontractility of ED caused by cavernous nerve injury human cavernous tissue by a mechanism involving Cavernous nerve injury is another common cause of overactivity of PKC. ED. Following cavernous nerve injury, animals show penile fibrosis, that is, decreased smooth muscle and increased collagen content.122 Gratzke et al.82 Hypertension-related ED reported that bilateral cavernous nerve injury causes Similar to diabetic ED, hypertension-related ED may increased RhoA and ROCK2 protein expression, and be related to augmented contractile signaling in the increased RhoA GTPase and ROCK activity in rat CC as well. Deoxycorticosterone acetate-salt-in- CC. The penile ROCK1 protein expression is un- duced hypertensive animals showed increased changed in these animals. However, Cho et al.123 cavernous contractile responses to both ET-1 and found increased ROCK1 expression in the CC of rats the a-adrenergic receptor agonist phenylephr- following cavernous nerve injury. In accordance ine.113,114 As to the downstream signaling, although with these findings, ICI of Y-27632 causes a protein expression levels of ROCK in the CC of significantly greater increase in intracavernosal deoxycorticosterone acetate-treated animals were pressure in nerve-injured rats compared to that similar to those of control animals, the phosphory- in sham-operated rats.123 lated form of myosin phosphatase regulatory subunit, a downstream effector of ROCK, was increased in cavernous tissue from deoxycorticosterone acet- 114 Inhibition of vasoconstriction to induce ate animals. In the spontaneously hypertensive erection rat, Wilkes et al.115 found that hypertension-related ED is associated with elevated penile RhoA levels Endogenous mechanisms and that inhibition of ROCK activity with Y-27632 The main driving force for penile erection is was beneficial in attenuating the decline in erectile NO-mediated signaling. Sexual stimulation or function. In another study, ROCK2 protein was nocturnal tumescence activates neuronal NO found to be elevated in spontaneous hypertensive synthase-mediated NO release from non-adrenergic rats.81 However, Behr-Roussel et al.116 found both a non-cholinergic nerve endings8,124 initiating vaso- reduced cavernous contractile response to phenyle- dilation. Subsequently, maintenance of dilation pherine and an impaired endothelium-dependent has been proposed to occur through sheer flow- relaxation to acetylcholine in spontaneous hyper- induced endothelial NOS activation.125 In the tensive rats. The authors proposed that the patho- smooth muscle cell, NO stimulates soluble guany- physiology of ED in hypertension is the result of an late cyclase, activating cGMP-dependent protein increase in cyclooxygenase-dependent constrictor kinase for cavernosal relaxation.126 tone, although a defect of endothelial or neuronal NO signaling was originally thought to induce NO production and/or bioavailability cannot be vasodilation by modulation of [Ca2 þ ] , through excluded. i 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 Aging-related ED Na þ –Ca2 þ exchangers activity and the activation Aging is closely related to ED, and interestingly, of sarco(endo)plasmic reticulum Ca2 þ -ATP despite an increased contractile response in the ases.56,127,128 It is now clear that NO signaling can CC,117 aged animals showed decreased (rather than also inhibit Ca2 þ -sensitizing mechanisms directly increased) penile concentration of NE110 and resulting in vasorelaxation. cGMP-dependent pro- expression of L-type VGCC and ryanodine recep- tein kinase can decrease levels of phosphorylated tors.118 It is possible that aging mainly impairs the MLC through telokin-mediated activation of MLC relaxation signaling, resulting in ‘unmasking’ of the phosphatase,93 as well as direct inhibition of RhoA contractile signaling. However, Jin et al.119 reported through its phosphorylation.129,130 Recombinant that though the expression of total RhoA remains cGMP-dependent protein kinase has been shown unchanged, membrane-bound RhoA and phos- 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/ that chronic administration of ET receptor antago- ROCK activity.129,130 It is tempting to speculate that nists might be beneficial in the protection of erectile decreased NO bioavailability leads to an increase in tissue. RhoA/ROCK constrictor activity. Elevated ROCK Ang-II: ACE inhibitors and Ang-II receptor block- activity may then mediate the increased vasocon- ers are widely used antihypertensive drugs. Since strictor sensitivity seen in various animal models of blockage of the Ang-II pathway results in smooth 2 þ ED. Endogenously, decreased [Ca ]i induced by muscle relaxation, these agents might be beneficial NO may also lead to vasodilation through inactiva- in the treatment of ED. Animal studies showed that tion of PKC, rapid CPI-17 dephosphorylation as well Ang-II receptor antagonists improved penile func- as MLCK inactivation, resulting in rapid MLC tion in aged animals or animals with dyslipide- dephosphorylation and relaxation.131 mia.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 Pharmacological therapies suggested that treatment with Ang-II receptor block- Blockage of upstream signaling (NE, ET-1, ANG-II). ers or ACE inhibitors are associated with improved NE: The non-selective a-adrenoceptor blocker, erectile function and sexual performance in patients phentolamine, can block the effect of NE by with hypertension and diabetes mellitus.144–146 competitively binding to the a1-adrenoceptor in However, a recent double-blind, randomized study, the CC, leading to the relaxation of the CSM.132 In involving 1549 patients, failed to reveal any sig- addition to the blockage of a-adrenoceptors, phen- nificant effect of an Ang-II receptor blockers or an tolamine may also induce relaxation of cavernous ACE inhibitor on ED.147 tissue by blocking ET-1 signaling.133 However, ICI with 5 mg phentolamine only resulted in penile Blockage of the accumulation of [Ca2 þ ] . Given tumescence, but not rigidity in humans.5 Currently, i that [Ca2 þ ] plays a central role in mediating CSM phentolamine is administered in conjunction with i contraction, drugs that inhibit the increase in other vasoactive agents, such as papaverine, pros- [Ca2 þ ] may be potentially effective in the treatment taglandin E1 and vasoactive intestinal polypeptide5 i of ED. This is supported by the findings that calcium as ICI therapies, with the advantages of lowering channel blockers (CCBs) significantly relaxed rab- the dosages and therefore the adverse effects of the bit48 and human148,149 CSM contracted by a-adre- other agents.134 The safety and efficacy of oral nergic agonists in vitro. However, ICI of CCBs in phentolamine in the treatment of ED have been dogs was less effective and had more side effects explored.135,136 Compared to sildenafil, oral phen- compared to papaverine.150 The results of clinical tolamine has a higher incidence of adverse effects trials with oral CCBs in the treatment of ED are and is less effective in improving penile erection.136 disappointing. CCBs exert either no effect or a Selective a1-adrenoceptor antagonists are com- negative effect on erectile function.151,152 The safety monly used to treat lower urinary tract symptoms. and efficacy of ICI of CCBs have also been Studies showed that they slightly improved erec- studied.153,154 Although the side effect of ICI of tile function as well.137,138 An additive effect was CCBs was comparable to other agents, CCBs may not observed when the selective a1-adrenoceptor anta- be as effective as blockade of a-adrenocep- gonist alfuzosin was used together with sildenafil to tors.148,153,154 treat ED.139 ET-1:AnETA receptor antagonist was shown to increase the duration of nerve-stimulated elevations Blockage of Ca2 þ -sensitizing pathway (mainly in intracavernosal pressure in rabbits, although the RhoA/ROCK). Ten years ago, the inhibition of peak pressure values were not altered.6 Despite the ROCK by ICI of Y-27632 was found to induce an 7 efficacy of the ETA receptor antagonist in animal erectile response in rats. This effect did not require studies, oral administration of this drug to men with activation of NO-mediated signaling, thus introdu- mild-to-moderate ED did not significantly improve cing a potential target pathway for the treatment of erectile function compared to placebo.6 This dis- ED, with potential extended benefit in cases where parity in efficacy between lab studies in rabbits and NO bioavailability was attenuated and thus phos- clinical studies was possibly due to important phodiesterase type 5 (PDE5) inhibitors were less differences between species with regard to the role efficacious. At this point, ROCK inhibitors are being of ET-1 in erectile function. This is further sup- developed and tested for a wide range of potential ported by the findings in rats that ET receptor uses. In Japan, the ROCK inhibitor fasudil has been antagonists do not significantly alter the erectile used to treat vasospasm following subarachnoid response, although they inhibit potent contractions hemorrhage155 and pulmonary hypertension.156 One to exogenous ET-1.140 Nevertheless, treatment with animal study showed that chronic administration of ET receptor antagonists for 2 weeks reversed oral fasudil prevented the development of ED cavernous apoptosis in diabetic rats,141 indicating and pelvic atherosclerosis.157 Another study using

International Journal of Impotence Research 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 Animal studies Human studies

ICI Systemic ICI Oral a-Adrenoceptor antagonists m ICP — Tumescence; used in Mildly improve adjunction with erection other agents 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 — Non-efficacious animals Calcium channel blockers Less effective than — Non-efficacious Non-efficacious papaverine 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 re- the need for intact NO-dependent signaling, is stores erectile function by suppressing corporal especially appealing for the treatment of ED asso- apoptosis caused by diabetes.74 These promising ciated with co-morbidities such as diabetes or results from animal studies indicate the possibilities prostatectomy, where NO signaling may be im- of administration of ROCK inhibitors either acutely paired. However, more than 10 years after the to induce erection or chronically to prevent or discovery of potent contractile signaling pathways reverse ED. To date, the effect of ROCK inhibitors in the cavernosum, we remain far from such targeted in the treatment of ED in humans has not been therapeutics. The most promise for therapeutic reported. In the era of oral therapy for ED, ICI intervention has been found with the inhibitor of therapy becomes less attractive. However, one ROCK signaling. The availability of the ROCK potential problem with systemic administration of inhibitor fasudil in Japan for the treatment of other ROCK inhibitors is whether it would cause pro- conditions makes clinical studies of its efficacy and found extra-cavernosous effects, such as hypoten- safety in the treatment of ED possible. Furthermore, sion. Further, the two ROCK isoforms share B90% isoform-selective ROCK inhibitors may be indicated homology in their kinase domains.158 This has made in the treatment of ED with different etiologies with it quite challenging to develop isoform-selective a reduced systemic side effect, although various inhibitors for clinical use. A recent compound, obstacles, such as isoform homology and lack of SLx2119 (Surface Logix), is unique in its specificity tissue specificity, have hindered their development. for only ROCK2.159 Other potential targets in both Ca2 þ -dependent A summary of the outcomes of both animal and and Ca2 þ -sensitizing signaling pathways may be human studies on the pharmacological targets mod- explored for benefits in the treatment of ED. ulating CSM contraction for the treatment of ED is Combined blockages of multiple targets in these shown in Table 1. The most significant advances pathways may enhance the efficacy in promoting during the past decade are listed as follows: penile erection. Development of these targeted therapeutics, which may benefit even cohorts a -Adrenoceptor antagonists have an additive refractory to PDE5 inhibition, are certainly promis- effect when administered together with other ED ing and warranted. medications. Chronic administration of ET receptor antagonists is beneficial in the protection of erectile tissue in rats. However, data in humans are lacking. Conflict of interest Chronic administration of ROCK inhibitors prevents ED or restores erectile function in animals. The authors declare no conflict of interest. However, clinical studies are needed.

References Conclusions This review outlines contractile signaling pathways, 1 NIH releases consensus statement on impotence. Am Fam Physician 1993; 48: 147–150. which play a role in the maintenance of penile 2 Andersson KE. Erectile physiological and pathophysiological flaccidity. The potential for therapeutic intervention pathways involved in erectile dysfunction. J Urol 2003; 170: at the level of smooth muscle contraction, bypassing S6–13.

International Journal of Impotence Research Inhibition of smooth muscle tone as a treatment for ED X Jiang and K Chitaley 56 3 Chitaley K. Type 1 and type 2 diabetic-erectile dysfunction: endothelin-1 in the rabbit corpus cavernosum. In Vivo 2006; same diagnosis (ICD-9), different disease? J Sex Med 2009; 20: 243–246. 6(Suppl 3): 262–268. 23 Parkkisenniemi UM, Klinge E. Functional characterization of 4 Chang S, Hypolite JA, Changolkar A, Wein AJ, Chacko S, endothelin receptors in the bovine retractor penis muscle Disanto ME. Increased contractility of diabetic rabbit corpora and penile artery. Pharmacol Toxicol 1996; 79: 73–79. smooth muscle in response to endothelin is medi- 24 Abeysinghe HR, Clancy J, Qiu Y. Comparison of endothelin- ated via Rho-kinase beta. Int J Impot Res 2003; 15: 53–62. 1-mediated tissue tension and calcium mobilization effects 5 Blum MD, Bahnson RR, Porter TN, Carter MF. Effect of in isolated rabbit corpus cavernosum. Urology 2002; 60: local alpha-adrenergic blockade on human penile erection. 925–930. J Urol 1985; 134: 479–481. 25 Sullivan ME, Dashwood MR, Thompson CS, Mikhailidis DP, 6 Kim NN, Dhir V, Azadzoi KM, Traish AM, Flaherty E, Morgan RJ. Down-regulation of endothelin-B receptor Goldstein I. Pilot study of the endothelin-A receptor selective sites in cavernosal tissue of hypercholesterolaemic rabbits. antagonist BMS-193884 for the treatment of erectile dysfunc- Br J Urol 1998; 81: 128–134. tion. J Androl 2002; 23: 76–83. 26 Sullivan ME, Dashwood MR, Thompson CS, Muddle JR, 7 Chitaley K, Wingard CJ, Clinton WR, Branam H, Stopper VS, Mikhailidis DP, Morgan RJ. Alterations in endothelin B Lewis RW et al. Antagonism of Rho-kinase stimulates rat receptor sites in cavernosal tissue of diabetic rabbits: penile erection via a nitric oxide-independent pathway. Nat potential relevance to the pathogenesis of erectile dysfunc- Med 2001; 7: 119–122. tion. J Urol 1997; 158: 1966–1972. 8 Saenz DTI, Blanco R, Goldstein I, Azadzoi K, De Las MA, 27 Uckert S, Fuhlenriede MH, Becker AJ, Stief CG, Scheller F, Krane RJ et al. Cholinergic neurotransmission in human Knapp WH et al. Is serotonin significant for the control corpus cavernosum. I. Responses of isolated tissue. Am J of penile flaccidity and detumescence in the human male? Physiol 1988; 254: H459–H467. Urol Res 2003; 31: 55–60. 9 Saenz DTI, Moroukian P, Tessier J, Kim JJ, Goldstein I, 28 Lau DH, Thompson CS, Bellringer JF, Thomas PJ, Frohrib D. Trabecular smooth muscle modulates the capa- Mumtaz FH, Morgan RJ et al. Doxazosin and serotonin citor function of the penis. Studies on a rabbit model. Am J (5-HT) receptor (1A, 2A, and 4) antagonists inhibit 5-HT- Physiol 1991; 260: H1590–H1595. mediated human cavernosal contraction. J Androl 2006; 27: 10 Chitaley K, Webb RC, Mills TM. Rhoa/Rho-kinase: a novel 679–685. player in the regulation of penile erection. Int J Impot Res 29 Furukawa K, Nagao K, Ishii N, Uchiyama T. Responses to 2001; 13: 67–72. serotonin (5HT) in isolated corpus cavernosum penis of 11 Takahashi R, Nishimura J, Hirano K, Naito S, Kanaide H. rabbit. Int J Impot Res 2003; 15: 267–271. Modulation of Ca2 þ sensitivity regulates contractility of 30 Iversen BM, Arendshorst WJ. Ang Ii and vasopressin rabbit corpus cavernosum smooth muscle. J Urol 2003; 169: stimulate calcium entry in dispersed smooth muscle 2412–2416. cells of preglomerular arterioles. Am J Physiol 1998; 274: 12 Sato M, Kawatani M. Effects of noradrenaline on cytosolic F498–F508. concentrations of Ca(2 þ ) in cultured corpus cavernosum 31 Jin L, Ying Z, Hilgers RH, Yin J, Zhao X, Imig JD et al. smooth muscle cells of the rabbit. Neurosci Lett 2002; 324: Increased RhoA/Rho-kinase signaling mediates spontaneous 89–92. tone in aorta from angiotensin Ii-induced hyper- 13 Holmquist F, Persson K, Garcia-Pascual A, Andersson KE. tensive rats. J Pharmacol Exp Ther 2006; 318: 288–295. Phospholipase C activation by endothelin-1 and noradrena- 32 Becker AJ, Uckert S, Stief CG, Scheller F, Knapp WH, line in isolated penile erectile tissue from rabbit. J Urol 1992; Hartmann U et al. Plasma levels of angiotensin II during 147: 1632–1635. different penile conditions in the cavernous and systemic 14 Murat N, Soner BC, Demir O, Esen A, Gidener S. Contractility blood of healthy men and patients with erectile dysfunction. of diabetic human corpus cavernosum smooth muscle in Urology 2001; 58: 805–810. response to serotonin mediated via Rho-kinase. Pharmacol- 33 Becker AJ, Uckert S, Stief CG, Truss MC, Machtens S, ogy 2009; 84: 24–28. Scheller F et al. Possible role of bradykinin and angiotensin 15 Rodat-Despoix L, Aires V, Ducret T, Marthan R, Savineau JP, Ii in the regulation of penile erection and detumescence. Rousseau E et al. Signalling pathways involved in the Urology 2001; 57: 193–198. contractile response to 5-HT in the human pulmonary artery. 34 Iwamoto Y, Song K, Takai S, Yamada M, Jin D, Sakaguchi M Eur Respir J 2009; 34: 1338–1347. et al. Multiple pathways of angiotensin I conversion and 16 Traish AM, Netsuwan N, Daley J, Padman-Nathan H, their functional role in the canine penile corpus cavernosum. Goldstein I, Saenz DTI. A heterogeneous population of alpha J Pharmacol Exp Ther 2001; 298: 43–48. 1 adrenergic receptors mediates contraction of human corpus 35 Yousif MH, Kehinde EO, Benter IF. Different responses to cavernosum smooth muscle to norepinephrine. angiotensin-(1–7) in young, aged and diabetic rabbit corpus J Urol 1995; 153: 222–227. cavernosum. Pharmacol Res 2007; 56: 209–216. 17 Saenz DTI, Kim N, Lagan I, Krane RJ, Goldstein I. 36 Kifor I, Williams GH, Vickers MA, Sullivan MP, Jodbert P, Regulation of adrenergic activity in penile corpus caverno- Dluhy RG. Tissue angiotensin II as a modulator of erec- sum. J Urol 1989; 142: 1117–1121. tile function. I. Angiotensin peptide content, secretion 18 Bonkowsky HL, Bloomer JR, Ebert PS, Mahoney MJ. and effects in the corpus cavernosum. J Urol 1997; 157: Heme synthetase deficiency in human protoporphyria. 1920–1925. demonstration of the defect in liver and cultured skin 37 Berridge MJ. Smooth muscle cell calcium activation mechan- fibroblasts. J Clin Invest 1975; 56: 1139–1148. isms. J Physiol 2008; 586: 5047–5061. 19 Saenz DTI, Carson MP, De Las MA, Goldstein I, Traish AM. 38 Bolton TB. Calcium events in smooth muscles and their Endothelin: localization, synthesis, activity, and receptor interstitial cells; physiological roles of sparks. J Physiol 2006; types in human penile corpus cavernosum. Am J Physiol 570: 5–11. 1991; 261: H1078–H1085. 39 House SJ, Potier M, Bisaillon J, Singer HA, Trebak M. The 20 Ortega MA, De Artinano AA. Highlights on endothelins: a non-excitable smooth muscle: calcium signaling and pheno- review. Pharmacol Res 1997; 36: 339–351. typic switching during vascular disease. Pflugers Arch 2008; 21 Dai Y, Pollock DM, Lewis RL, Wingard CJ, Stopper VS, Mills 456: 769–785. TM. Receptor-specific influence of endothelin-1 in the 40 Gomez MF, Stevenson AS, Bonev AD, Hill-Eubanks DC, erectile response of the rat. Am J Physiol Regul Integr Comp Nelson MT. Opposing actions of inositol 1,4,5-trisphosphate Physiol 2000; 279: R25–R30. and ryanodine receptors on nuclear factor of activated 22 Mumtaz FH, Lau DH, Siddiqui EJ, Thompson CS, T-cells regulation in smooth muscle. J Biol Chem 2002; 277: Morgan RJ, Mikhailidis DP. Pharmacological properties of 37756–37764.

International Journal of Impotence Research Inhibition of smooth muscle tone as a treatment for ED X Jiang and K Chitaley 57 41 Dreja K, Hellstrand P. Differential modulation of caffeine- urinary tract tissue enzymes. Mol Cell Biochem 2007; 301: and IP3-induced calcium release in cultured arterial tissue. 227–233. Am J Physiol 1999; 276: C1115–C1120. 60 Disanto ME, Wang Z, Menon C, Zheng Y, Chacko T, Hypolite 42 Zhou H, Nakamura T, Matsumoto N, Hisatsune C, Mizutani J et al. Expression of myosin isoforms in smooth muscle cells A, Iesaki T et al. Predominant role of type 1 IP3 receptor in in the corpus cavernosum penis. Am J Physiol 1998; 275: aortic vascular muscle contraction. Biochem Biophys Res C976–C987. Commun 2008; 369: 213–219. 61 Zhang XH, Melman A, Disanto ME. Update on corpus 43 Liu X, Farley JM. Acetylcholine-induced Ca þþ-dependent cavernosum smooth muscle contractile pathways in chloride current oscillations are mediated by inositol 1,4,5- erectile function: a role for testosterone? J Sex Med 2011; 8: trisphosphate in tracheal myocytes. J Pharmacol Exp Ther 1865–1879. 1996; 277: 796–804. 62 Kitazawa T, Masuo M, Somlyo AP. G protein-mediated 44 Essin K, Gollasch M. Role of ryanodine receptor subtypes in inhibition of myosin light-chain phosphatase in vascular initiation and formation of calcium sparks in arterial smooth smooth muscle. Proc Natl Acad Sci USA 1991; 88: muscle: comparison with striated muscle. J Biomed Biotech- 9307–9310. nol 2009; 2009: 135249. 63 Chuang AT, Strauss JD, Murphy RA, Steers WD. Sildenafil, a 45 Williams BA, Sims SM. Calcium sparks activate calcium- type-5 cGMP phosphodiesterase inhibitor, specifically dependent ClÀ current in rat corpus cavernosum amplifies endogenous cGMP-dependent relaxation in rabbit smooth muscle cells. Am J Physiol Cell Physiol 2007; 293: corpus cavernosum smooth muscle in vitro. J Urol 1998; 160: C1239–C1251. 257–261. 46 Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot 64 Wu X, Somlyo AV, Somlyo AP. Cyclic GMP-dependent HJ et al. Relaxation of arterial smooth muscle by calcium stimulation reverses G-protein-coupled inhibition of smooth sparks. Science 1995; 270: 633–637. muscle myosin light chain phosphate. Biochem Biophys Res 47 Mccloskey C, Cagney V, Large R, Hollywood M, Sergeant G, Commun 1996; 220: 658–663. Mchale N et al. Voltage-dependent Ca2 þ currents contribute 65 Pfitzer G, Arner A. Involvement of small GTPases in the to spontaneous Ca2 þ waves in rabbit corpus cavernosum regulation of smooth muscle contraction. Acta Physiol Scand myocytes. J Sex Med 2009; 6: 3019–3031. 1998; 164: 449–456. 48 Kerfoot WW, Park HY, Schwartz LB, Hagen PO, Carson III CC. 66 Taggart MJ, Lee YH, Morgan KG. Cellular redistribution of Characterization of calcium channel blocker induced smooth PKCalpha, rhoA, and ROKalpha following smooth muscle muscle relaxation using a model of isolated corpus caverno- agonist stimulation. Exp Cell Res 1999; 251: 92–101. sum. J Urol 1993; 150: 249–252. 67 Ohkawara H, Ishibashi T, Sakamoto T, Sugimoto K, 49 Craven M, Sergeant GP, Hollywood MA, Mchale NG, Nagata K, Yokoyama K et al. Thrombin-induced rapid Thornbury KD. Modulation of spontaneous Ca2 þ -activated geranylgeranylation of RhoA as an essential process for ClÀ currents in the rabbit corpus cavernosum by the nitric RhoA activation in endothelial cells. J Biol Chem 2005; 280: oxide-cGMP pathway. J Physiol 2004; 556: 495–506. 10182–10188. 50 Yanai Y, Hashitani H, Kubota Y, Sasaki S, Kohri K, 68 Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Suzuki H. The role of Ni(2 þ )-sensitive T-type Ca(2 þ ) Nakafuku M et al. Regulation of myosin phosphatase by channels in the regulation of spontaneous excitation in Rho and Rho-associated kinase (Rho-kinase). Science 1996; detrusor smooth muscles of the guinea-pig bladder. BJU Int 273: 245–248. 2006; 97: 182–189. 69 Somlyo AP, Somlyo AV. Signal transduction through the 51 Zhong J, Hume JR, Keef KD. Beta-adrenergic receptor RhoA/Rho-kinase pathway in smooth muscle. J Muscle Res stimulation of L-type Ca2 þ channels in rabbit portal vein Cell Motil 2004; 25: 613–615. myocytes involves both alphas and betagamma G protein 70 Amano M, Ito M, Kimura K, Fukata Y, Chihara K, Nakano T subunits. J Physiol 2001; 531: 105–115. et al. Phosphorylation and activation of myosin by 52 Bray JG, Mynlieff M. Involvement of protein kinase C and Rho-associated kinase (Rho-kinase). J Biol Chem 1996; 271: protein kinase A in the enhancement of L-type calcium 20246–20249. current by GABAB receptor activation in neonatal hippo- 71 Teixeira CE, Jin L, Ying Z, Palmer T, Priviero FB, Webb RC. campus. Neuroscience 2011; 179: 62–72. Expression and functional role of the RhoA/Rho-kinase 53 Mcneish AJ, Altayo FJ, Garland CJ. Evidence both L-type and pathway in rat coeliac artery. Clin Exp Pharmacol Physiol non-L-type voltage-dependent calcium channels contribute 2005; 32: 817–824. to cerebral artery vasospasm following loss of no in the rat. 72 Ark M, Ozveren E, Yazici G, Korkmaz B, Buyukafsar K, Vascul Pharmacol 2010; 53: 151–159. Arikan O et al. Effects of HA-1077 and Y-27632, two Rho- 54 Yuill KH, Mcneish AJ, Kansui Y, Garland CJ, Dora KA. kinase inhibitors, in the human umbilical artery. Cell Nitric oxide suppresses cerebral vasomotion by sGC-inde- Biochem Biophys 2004; 41: 331–342. pendent effects on ryanodine receptors and voltage-gated 73 Nakamura K, Nishimura J, Hirano K, Ibayashi S, Fujishima calcium channels. J Vasc Res 2010; 47: 93–107. M, Kanaide H. Hydroxyfasudil, an active metabolite of 55 Pritchard TJ, Bowman PS, Jefferson A, Tosun M, Lynch RM, fasudil hydrochloride, relaxes the rabbit basilar artery by Paul RJ. Na( þ )–K( þ )-atpase and Ca(2 þ ) clearance proteins disinhibition of myosin light chain phosphatase. J Cereb in smooth muscle: a functional unit. Am J Physiol Heart Circ Blood Flow Metab 2001; 21: 876–885. Physiol 2010; 299: H548–H556. 74 Li WJ, Park K, Paick JS, Kim SW. Chronic treatment with an 56 Cohen RA, Weisbrod RM, Gericke M, Yaghoubi M, Bierl C, oral Rho-kinase inhibitor restores erectile function by Bolotina VM. Mechanism of nitric oxide-induced vasodilata- suppressing corporal apoptosis in diabetic rats. J Sex Med tion: refilling of intracellular stores by sarcoplasmic reticu- 2011; 8: 400–410. lum Ca2 þ ATPase and inhibition of store-operated Ca2 þ 75 Ishizaki T, Maekawa M, Fujisawa K, Okawa K, Iwamatsu A, influx. Circ Res 1999; 84: 210–219. Fujita A et al. The small GTP-binding protein Rho binds 57 Furukawa K, Ohshima N, Tawada-Iwata Y, Shigekawa M. to and activates a 160 kDA Ser/Thr protein kinase homo- Cyclic GMP stimulates Na þ /Ca2 þ exchange in vascular logous to myotonic dystrophy kinase. EMBO J 1996; 15: smooth muscle cells in primary culture. J Biol Chem 1991; 1885–1893. 266: 12337–12341. 76 Takahashi N, Tuiki H, Saya H, Kaibuchi K. Localization of the 58 Cartwright EJ, Oceandy D, Neyses L. Plasma membrane gene coding for ROCK II/Rho kinase on human chromosome calcium ATPase and its relationship to nitric oxide signaling 2p24. Genomics 1999; 55: 235–237. in the heart. Ann NY Acad Sci 2007; 1099: 247–253. 77 Satoh KMD, Fukumoto Y, Shimokawa H. Rho-kinase: 59 Juan YS, Onal B, Broadaway S, Cosgrove J, Leggett RE, important new therapeutic target in cardiovascular diseases. Whitbeck C et al. Effect of castration on male rabbit lower Am J Physiol Heart Circ Physiol 2011; 301: H287–H296.

International Journal of Impotence Research Inhibition of smooth muscle tone as a treatment for ED X Jiang and K Chitaley 58 78 Wang Y, Zheng XR, Riddick N, Bryden M, Baur W, Zhang X 96 Bousquet SM, Monet M, Boulay G. Protein kinase C- et al. Rock isoform regulation of myosin phosphatase and dependent phosphorylation of transient receptor potential contractility in vascular smooth muscle cells. Circ Res 2009; canonical 6 (TRPC6) on serine 448 causes channel inhibition. 104: 531–540. J Biol Chem 2010; 285: 40534–40543. 79 Wang H, Eto M, Steers WD, Somlyo AP, Somlyo AV. 97 Paffett ML, Riddle MA, Kanagy NL, Resta TC, Walker BR. RhoA-mediated Ca2 þ sensitization in erectile function. Altered protein kinase c regulation of pulmonary endothelial J Biol Chem 2002; 277: 30614–30621. store- and receptor-operated Ca2 þ entry after chronic 80 Chang S, Hypolite JA, Zderic SA, Wein AJ, Chacko S, Disanto hypoxia. J Pharmacol Exp Ther 2010; 334: 753–760. ME. Increased corpus cavernosum smooth muscle tone 98 Kline LW, Ji J, Wang GJ, Sutherland SK, Pang PK, Benishin associated with partial bladder outlet obstruction is mediated CG. Protein kinase C masks nitric oxide synthase activity in via Rho-kinase. Am J Physiol Regul Integr Comp Physiol vascular smooth muscle under basal conditions. J Cardiovasc 2005; 289: R1124–R1130. Pharmacol 2004; 43: 281–287. 81 Zhu PY, Jiang R, Deng QF, Wang XR. Expression of Rho- 99 Jiang X, Yang F, Tan H, Liao D, Bryan Jr RM, Randhawa JK kinase and heme oxygenase in the corpus cavernosum of et al. Hyperhomocystinemia impairs endothelial function spontaneous hypertensive rats. Zhonghua Nan Ke Xue 2008; and eNOS activity via PKC activation. Arterioscler Thromb 14: 215–219. Vasc Biol 2005; 25: 2515–2521. 82 Gratzke C, Strong TD, Gebska MA, Champion HC, Stief CG, 100 Muniyappa R, Srinivas PR, Ram JL, Walsh MF, Sowers JR. Burnett AL et al. Activated RhoA/Rho kinase impairs erectile Calcium and protein kinase C mediate high-glucose-induced function after cavernous nerve injury in rats. J Urol 2010; 184: inhibition of inducible nitric oxide synthase in vascular 2197–2204. smooth muscle cells. Hypertension 1998; 31: 289–295. 83 Vignozzi L, Morelli A, Filippi S, Ambrosini S, Mancina R, 101 Angulo J, Cuevas P, Fernandez A, Allona A, Moncada I, Luconi M et al. Testosterone regulates RhoA/Rho-kinase Martin-Morales A et al. Enhanced thromboxane receptor- signaling in two distinct animal models of chemical diabetes. mediated responses and impaired endothelium-dependent J Sex Med 2007; 4: 620–630. relaxation in human corpus cavernosum from diabetic 84 Chiou WF, Liu HK, Juan CW. Abnormal protein expression in impotent men: role of protein kinase C activity. J Pharmacol the corpus cavernosum impairs erectile function in type 2 Exp Ther 2006; 319: 783–789. diabetes. BJU Int 2010; 105: 674–680. 102 Jin L, Teixeira CE, Webb RC, Leite R. Comparison of 85 Shcherbakova OV, Serebryanaya DV, Postnikov AB, the involvement of protein kinase C in agonist-induced Schroeter MM, Zittrich S, Noegel AA et al. Kinase-related contractions in mouse aorta and corpus cavernosum. protein/telokin inhibits Ca2 þ -independent contraction in Eur J Pharmacol 2008; 590: 363–368. Triton-skinned guinea pig Taenia coli. Biochem J 2010; 429: 103 Eto M, Senba S, Morita F, Yazawa M. Molecular cloning 291–302. of a novel phosphorylation-dependent inhibitory protein 86 Herring BP, Lyons GE, Hoggatt AM, Gallagher PJ. Telokin of protein phosphatase-1 (CPI17) in smooth muscle: its expression is restricted to smooth muscle tissues during specific localization in smooth muscle. FEBS Lett 1997; mouse development. Am J Physiol Cell Physiol 2001; 280: 410: 356–360. C12–C21. 104 Kitazawa T, Eto M, Woodsome TP, Khalequzzaman M. 87 Sobieszek A, Andruchov OY, Nieznanski K. Kinase-related Phosphorylation of the myosin phosphatase targeting sub- protein (Telokin) is phosphorylated by smooth-muscle unit and CPI-17 during Ca2 þ sensitization in rabbit smooth myosin light-chain kinase and modulates the kinase activity. muscle. J Physiol 2003; 546: 879–889. Biochem J 1997; 328(Part 2): 425–430. 105 Koyama M, Ito M, Feng J, Seko T, Shiraki K, Takase K et al. 88 Nieznanski K, Sobieszek A. Telokin (kinase-related protein) Phosphorylation of CPI-17, an inhibitory phosphoprotein modulates the oligomeric state of smooth-muscle myosin of smooth muscle myosin phosphatase, by Rho-kinase. FEBS light-chain kinase and its interaction with myosin filaments. Lett 2000; 475: 197–200. Biochem J 1997; 322(Part 1): 65–71. 106 Takizawa N, Koga Y, Ikebe M. Phosphorylation of CPI17 and 89 Numata T, Katoh T, Yazawa M. Functional role of the myosin binding subunit of type 1 protein phosphatase by C-terminal domain of smooth muscle myosin light chain p21-activated kinase. Biochem Biophys Res Commun 2002; kinase on the phosphorylation of smooth muscle myosin. 297: 773–778. J Biochem 2001; 129: 437–444. 107 Kolitsi Z, Panayiotakis G, Anastassopoulos V, Scodras A, 90 Shirinsky VP, Vorotnikov AV, Birukov KG, Nanaev AK, Pallikarakis N. A multiple projection method for digital Collinge M, Lukas TJ et al. A kinase-related protein stabilizes tomosynthesis. Med Phys 1992; 19: 1045–1050. unphosphorylated smooth muscle myosin minifilaments in 108 Somlyo AP, Somlyo AV. Ca2 þ sensitivity of smooth the presence of ATP. J Biol Chem 1993; 268: 16578–16583. muscle and nonmuscle myosin II: modulated by G proteins, 91 Choudhury N, Khromov AS, Somlyo AP, Somlyo AV. kinases, and myosin phosphatase. Physiol Rev 2003; 83: Telokin mediates Ca2 þ -desensitization through activation 1325–1358. of myosin phosphatase in phasic and tonic smooth muscle. 109 Chang S, Hypolite JA, Mohanan S, Zderic SA, Wein AJ, J Muscle Res Cell Motil 2004; 25: 657–665. Chacko S. Alteration of the PKC-mediated signaling pathway 92 Walker LA, Macdonald JA, Liu X, Nakamoto RK, Haystead TA, for smooth muscle contraction in obstruction-induced Somlyo AV et al. Site-specific phosphorylation and point hypertrophy of the urinary bladder. Lab Invest 2009; 89: mutations of Telokin modulate its Ca2 þ -desensitizing effect in 823–832. smooth muscle. J Biol Chem 2001; 276: 24519–24524. 110 Morrison JF, Pallot DJ, Sheen R, Dhanasekaran S, 93 Wu X, Haystead TA, Nakamoto RK, Somlyo AV, Somlyo AP. Mensah-Brown EP. The effects of age and streptozotocin Acceleration of myosin light chain dephosphorylation and diabetes on the sympathetic innervation in the rat penis. relaxation of smooth muscle by telokin. Synergism with Mol Cell Biochem 2007; 295: 53–58. cyclic nucleotide-activated kinase. J Biol Chem 1998; 273: 111 Takahashi K, Ghatei MA, Lam HC, O’halloran DJ, Bloom SR. 11362–11369. Elevated plasma endothelin in patients with diabetes 94 Khromov AS, Wang H, Choudhury N, Mcduffie M, Herring mellitus. Diabetologia 1990; 33: 306–310. BP, Nakamoto R et al. Smooth muscle of telokin-deficient 112 Haak T, Jungmann E, Felber A, Hillmann U, Usadel KH. mice exhibits increased sensitivity to Ca2 þ and decreased Increased plasma levels of endothelin in diabetic patients cGMP-induced relaxation. Proc Natl Acad Sci USA 2006; with hypertension. Am J Hypertens 1992; 5: 161–166. 103: 2440–2445. 113 Carneiro FS, Giachini FR, Lima VV, Carneiro ZN, Nunes KP, 95 Crozatier B. Central role of PKCs in vascular smooth Ergul A et al. DOCA-salt treatment enhances responses to muscle cell ion channel regulation. J Mol Cell Cardiol 2006; endothelin-1 in murine corpus cavernosum. Can J Physiol 41: 952–955. Pharmacol 2008; 86: 320–328.

International Journal of Impotence Research Inhibition of smooth muscle tone as a treatment for ED X Jiang and K Chitaley 59 114 Carneiro FS, Nunes KP, Giachini FR, Lima VV, Carneiro ZN, dysfunction: experience with 100 patients. J Urol 1986; 135: Nogueira EF et al. Activation of the ET-1/ETA pathway 704–706. contributes to erectile dysfunction associated with miner- 133 Traish A, Gupta S, Gallant C, Huang YH, Goldstein I. alocorticoid hypertension. J Sex Med 2008; 5: 2793–2807. Phentolamine mesylate relaxes penile corpus cavernosum 115 Wilkes N, White S, Stein P, Bernie J, Rajasekaran M. tissue by adrenergic and non-adrenergic mechanisms. Phosphodiesterase-5 inhibition synergizes Rho-kinase antag- Int J Impot Res 1998; 10: 215–223. onism and enhances erectile response in male hypertensive 134 Bella AJ, Brock GB. Intracavernous pharmacotherapy for rats. Int J Impot Res 2004; 16: 187–194. erectile dysfunction. Endocrine 2004; 23: 149–155. 116 Behr-Roussel D, Chamiot-Clerc P, Bernabe J, Mevel K, 135 Padma-Nathan H, Goldstein I, Klimberg I, Coogan C, Alexandre L, Safar ME et al. Erectile dysfunction Auerbach S, Lammers P. Long-term safety and efficacy of oral in spontaneously hypertensive rats: pathophysiological phentolamine mesylate (Vasomax) in men with mild to mechanisms. Am J Physiol Regul Integr Comp Physiol 2003; moderate erectile dysfunction. Int J Impot Res 2002; 14: 284: R682–R688. 266–270. 117 Christ GJ, Stone B, Melman A. Age-dependent alterations in 136 Ugarte F, Hurtado-Coll A. Comparison of the efficacy and the efficacy of phenylephrine-induced contractions in vas- safety of sildenafil citrate (Viagra) and oral phentolamine cular smooth muscle isolated from the corpus cavernosum of for the treatment of erectile dysfunction. Int J Impot Res impotent men. Can J Physiol Pharmacol 1991; 69: 909–913. 2002; 14(Suppl 2): S48–S53. 118 Luo Y, Jiang R. Expressions of Cav1.3 and RyR1 in the corpus 137 Lowe FC. Treatment of lower urinary tract symptoms cavernosum of the penis in aged rats. Zhonghua Nan Ke Xue suggestive of benign prostatic hyperplasia: sexual function. 2009; 15: 985–989. BJU Int 2005; 95(Suppl 4): 12–18. 119 Jin L, Liu T, Lagoda GA, Champion HC, Bivalacqua TJ, 138 Carson CC. Combination of phosphodiesterase-5 inhibitors Burnett AL. Elevated RhoA/Rho-kinase activity in the aged and alpha-blockers in patients with benign prostatic hyper- rat penis: mechanism for age-associated erectile dysfunction. plasia: treatments of lower urinary tract symptoms, erectile FASEB J 2006; 20: 536–538. dysfunction, or both? BJU Int 2006; 97(Suppl 2): 39–43. 120 Park K, Shin JW, Oh JK, Ryu KS, Kim SW, Paick JS. 139 Kaplan SA, Gonzalez RR, Te AE. Combination of alfuzosin Restoration of erectile capacity in normotensive aged rats and sildenafil is superior to monotherapy in treating by modulation of angiotensin receptor type 1. J Androl 2005; lower urinary tract symptoms and erectile dysfunction. 26: 123–128. Eur Urol 2007; 51: 1717–1723. 121 Rajasekaran M, White S, Baquir A, Wilkes N. Rho-kinase 140 Merlin SL, Brock GB, Begin LR, Hiou Tim FF, Macramalla inhibition improves erectile function in aging male AN, Seyam RM et al. New insights into the role of Brown–Norway rats. J Androl 2005; 26: 182–188. endothelin-1 in radiation-associated impotence. Int J Impot 122 Leungwattanakij S, Bivalacqua TJ, Usta MF, Yang DY, Res 2001; 13: 104–109. Hyun JS, Champion HC et al. Cavernous neurotomy causes 141 Jesmin S, Zaedi S, Yamaguchi N, Maeda S, Yamaguchi I, hypoxia and fibrosis in rat corpus cavernosum. J Androl Goto K et al. Effects of dual endothelin receptor antagonist on 2003; 24: 239–245. antiapoptotic marker Bcl-2 expression in streptozotocin- 123 Cho MC, Park K, Chai JS, Lee SH, Kim SW, Paick JS. induced diabetic rats. Exp Biol Med (Maywood) 2006; 231: Involvement of sphingosine-1-phosphate/RhoA/Rho-kinase 1034–1039. signaling pathway in corporal fibrosis following cavernous 142 Baumhakel M, Custodis F, Schlimmer N, Laufs U, Bohm M. nerve injury in male rats. J Sex Med 2010; 8: 712–721. Improvement of endothelial function of the corpus 124 Burnett AL, Nelson RJ, Calvin DC, Liu JX, Demas GE, cavernosum in apolipoprotein E knockout mice treated with Klein SL et al. Nitric oxide-dependent penile erection in irbesartan. J Pharmacol Exp Ther 2008; 327: 692–698. mice lacking neuronal nitric oxide synthase. Mol Med 1996; 143 Dorrance AM, Lewis RW, Mills TM. Captopril treat- 2: 288–296. ment reverses erectile dysfunction in male stroke prone 125 Hurt KJ, Musicki B, Palese MA, Crone JK, Becker RE, spontaneously hypertensive rats. Int J Impot Res 2002; 14: Moriarity JL et al. Akt-dependent phosphorylation of 494–497. endothelial nitric-oxide synthase mediates penile erection. 144 Fogari R, Zoppi A, Corradi L, Mugellini A, Poletti L, Proc Natl Acad Sci USA 2002; 99: 4061–4066. Lusardi P. Sexual function in hypertensive males treated 126 Burnett AL. Nitric oxide regulation of penile erection: with lisinopril or atenolol: a cross-over study. Am J biology and therapeutic implications. J Androl 2002; 23: Hypertens 1998; 11: 1244–1247. S20–S26. 145 Fogari R, Zoppi A, Poletti L, Marasi G, Mugellini A, 127 Plane F, Wiley KE, Jeremy JY, Cohen RA, Garland CJ. Corradi L. Sexual activity in hypertensive men treated with Evidence that different mechanisms underlie smooth muscle valsartan or carvedilol: a crossover study. Am J Hypertens relaxation to nitric oxide and nitric oxide donors in the 2001; 14: 27–31. rabbit isolated carotid artery. Br J Pharmacol 1998; 123: 146 Baumhakel M, Schlimmer N, Bohm M. Effect of irbesartan on 1351–1358. erectile function in patients with hypertension and meta- 128 Sanders DB, Kelley T, Larson D. The role of nitric oxide bolic syndrome. Int J Impot Res 2008; 20: 493–500. synthase/nitric oxide in vascular smooth muscle control. 147 Bohm M, Baumhakel M, Teo K, Sleight P, Probstfield J, Gao P Perfusion 2000; 15: 97–104. et al. Erectile dysfunction predicts cardiovascular events in 129 Sauzeau V, Le JH, Cario-Toumaniantz C, Smolenski A, high-risk patients receiving telmisartan, ramipril, or both: Lohmann SM, Bertoglio J et al. Cyclic GMP-dependent The ONgoing Telmisartan Alone and in combination with protein kinase signaling pathway inhibits RhoA-induced Ramipril Global Endpoint Trial/Telmisartan Randomized Ca2 þ sensitization of contraction in vascular smooth muscle. AssessmeNt Study in ACE iNtolerant subjects with cardio- J Biol Chem 2000; 275: 21722–21729. vascular Disease (ONTARGET/TRANSCEND) trials. Circula- 130 Sawada N, Itoh H, Yamashita J, Doi K, Inoue M, Masatsugu K tion 2010; 121: 1439–1446. et al. cGMP-dependent protein kinase phosphorylates and 148 Fovaeus M, Andersson KE, Hedlund H. Effects of some inactivates RhoA. Biochem Biophys Res Commun 2001; 280: calcium channel blockers on isolated human penile erectile 798–805. tissues. J Urol 1987; 138: 1267–1272. 131 Kitazawa T, Semba S, Huh YH, Kitazawa K, Eto M. 149 Ma LL, Liu YQ, Tang WH, Zhao LM, Jiang H. Experimental Nitric oxide-induced biphasic mechanism of vascular relaxa- study of verapamil on the relaxation of isolated human tion via dephosphorylation of CPI-17 and MYPT1. J Physiol corpus cavernosum tissues. Asian J Androl 2006; 8: 195–198. 2009; 587: 3587–3603. 150 Sarikaya S, Asci R, Aybek Z, Yilmaz AF, Buyukalpelli R, 132 Sidi AA, Cameron JS, Duffy LM, Lange PH. Intracavernous Yildiz S. Effects of intracavernous calcium channel blockers drug-induced erections in the management of male erectile in dogs. Int Urol Nephrol 1997; 29: 673–680.

International Journal of Impotence Research Inhibition of smooth muscle tone as a treatment for ED X Jiang and K Chitaley 60 151 Morrissette DL, Skinner MH, Hoffman BB, Levine RE, 155 Shibuya M, Suzuki Y. Treatment of cerebral vasospasm by Davidson JM. Effects of antihypertensive drugs atenolol and a protein kinase inhibitor at 877. No To Shinkei 1993; 45: nifedipine on sexual function in older men: a placebo- 819–824. controlled, crossover study. Arch Sex Behav 1993; 22: 156 Doggrell SA. Rho-kinase inhibitors show promise in 99–109. pulmonary hypertension. Expert Opin Invest Drugs 2005; 152 Fogelman J. Verapamil caused depression, confusion, and 14: 1157–1159. impotence. Am J Psychiatry 1988; 145: 380. 157 Park K, Kim SW, Rhu KS, Paick JS. Chronic administration of 153 Bolayir K, Goksin N. Intracavernous self-injection of an oral Rho kinase inhibitor prevents the development papaverine and verapamil: a clinical experience. Acta Chir of vasculogenic erectile dysfunction in a rat model. Hung 1994; 34: 253–256. J Sex Med 2006; 3: 996–1003. 154 Sahin M, Basar MM, Bozdogan O, Atan A. Short-term 158 Liao JK, Seto M, Noma K. Rho kinase (ROCK) inhibitors. histopathologic effects of different intracavernosal agents J Cardiovasc Pharmacol 2007; 50: 17–24. on corpus cavernosum and antifibrotic activity of intraca- 159 Shimokawa H, Rashid M. Development of Rho-kinase vernosal verapamil: an experimental study. Urology 2001; 58: inhibitors for cardiovascular medicine. Trends Pharmacol 487–492. Sci 2007; 28: 296–302.

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