International Journal of Impotence Research (2003) 15, Suppl 5, S20–S24 & 2003 Nature Publishing Group All rights reserved 0955-9930/03 $25.00 www.nature.com/ijir Vasoconstriction, RhoA/Rho-kinase and the erectile response

TM Mills1,2*, RW Lewis2, CJ Wingard1,2, AE Linder1, L Jin1 and RC Webb1,2

1Department of Physiology, Medical College of Georgia, Augusta, Georgia, USA; and 2Department of Surgery (Urology), Medical College of Georgia, Augusta, Georgia, USA

Recent studies have suggested that contraction of the smooth muscle in the cavernosal arterioles and in the walls of the cavernosal sinuses is maintained by the RhoA/Rho-kinase signaling pathway. However, this contraction activity must be overcome to permit the vasorelaxation essential for . We postulate that nitric oxide (NO) causes erection primarily by inhibiting the RhoA/Rho-kinase pathway. The following will discuss evidence in support of the important role of Rho-kinase-mediated vasoconstriction in the nonerect and how NO overrides this Rho- kinase-mediated vasoconstriction to permit vasodilation and erection. International Journal of Impotence Research (2003) 15, Suppl 5, S20–S24. doi:10.1038/sj.ijir.3901068

Keywords: penile erection; nitric oxide; Rho-kinase

Introduction which are lined with endothelial cells (sinusoidal endothelial cells) and contains smooth muscle (sinusoidal smooth muscle) in their walls. Blood is The majority of the time, the smooth muscle cells in conveyed out of the sinuses via the thin-walled the erectile tissue remain contracted and blood flow veins lying immediately beneath the tunica albugi- into the cavernous sinuses is minimal. Studies from nea (subtunical veins). The cavernosal arterioles several laboratories strongly support the concept also containing endothelial cells overlie layers of that RhoA/Rho-kinase-mediated vasoconstriction is smooth muscle cells. When the arteriolar smooth primarily responsible for keeping penile smooth muscle layer is contracted, only small volumes of 1–5 muscle in the contracted state. Nitric oxide (NO), blood are delivered to the sinuses and this volume is released by autonomic innervation and by endothe- readily drained out through the subtunical veins. lial cells leads to increased production of cGMP and Thus, the contraction of arteriolar and sinusoidal activation of protein kinase G (PKG). We have smooth muscle limits blood flow into the erectile proposed that the NO/cGMP/PKG pathway inhibits tissue and keeps the penis in the nonerect state. In RhoA/Rho-kinase-mediated vasoconstriction allow- order for erection to occur, the smooth muscle in the ing smooth muscle relaxation and the increased arterioles and cavernous sinuses must relax. As the blood flow required for erection. arterioles relax, blood flow into the cavernous sinuses increases and exert shear and stretch force on the sinusoidal endothelial cells. The endothelial Erectile physiology6 cells respond with the activation of eNOS activity and the release of additional NO which also contributes to smooth muscle relaxation. As the Erectile tissue is housed in the two dorsal corpora sinusoidal smooth muscle relaxes, the sinuses cavernosa and the single ventral corpus spongiosum expand against the tunica albuginea and blood of the penis. Since the spongiosum contributes little outflow decreases (veno-occlusion). The increased to the rigidity during erection, it will not be further inflow and decreased outflow result in the high discussed. The corpora cavernosa contain connec- intracavernosal pressures which are characteristics tive tissue arranged in a trabecular meshwork which of the erect penis. is continuous with the tunica albuginea. This meshwork limits the system of vascular sinuses VasoconstrictionFmaintenance of the penis in the flaccid state *Correspondence: TM Mills, Department of Physiology, Medical College of Georgia, Augusta, GA 30912-3000, USA. The following contains a brief review of the E-mail: [email protected] pharmacology of penile erection with emphasis Erectile response TM Mills et al S21 placed on the role of the RhoA/Rho-kinase calcium inhibiting L-type Ca2+ channels, (2) activating sensitization pathway. A more general description of Ca2+-dependent ATPase in the sarcoplasmic reticu- penile pharmacology can be found in several recent lum and/or (3) hyperpolarizing the cell membrane reviews.6–9 The penis is held in the nonerect state by causing increased opening of membrane K+ except during periods of sexual excitement and channels.20 during nocturnal penile events (non- sexual that occur during sleep). The contraction of penile smooth muscle appears to be mediated by the actions of a-adrenergic agonists and RhoA/Rho-kinase calcium sensitization pathway (Figure 1) by the actions of endothelin-1 acting via ETA 8,10,11 receptors. The physiology of the ETB receptors in penile erection remains less clear.12 In other 13 While there is ample conceptual support for the vascular systems, binding of ET-1 to the ETB importance of intracellular calcium in regulating receptors in the endothelial cells activates endothe- 13 vasoconstriction and vasodilation in the penile lial NO synthesis and smooth muscle relaxation, vasculature, Ca2+-sensitization has only recently but this has not been extensively studied in penile been investigated in the erectile response. In other tissue. vascular tissues, Ca2+-sensitization is, in part, The initial contraction of vascular smooth muscle dependent on enzymes of the RhoA/Rho-kinase depends on elevated intracellular levels of cytosolic 14,16,19 2+ pathway. RhoA is a small, GTP-binding free calcium ([Ca ]i) which occur when ligand protein that is involved in several cellular processes binding leads to the opening of membrane Ca2+ 2+ such as morphology, cytoskeletal function, secretion channels and the release of intracellular Ca 14 14 2+ and smooth muscle contraction. When RhoA is stores. The intracellular Ca complexes with bound to GDP, it is inactive, but with heterotrimeric calmodulin, the complex binds to myosin light G-protein activation (from ligand binding), the GDP chain kinase (MLCK) resulting in its activation. is exchanged for GTP. The RhoA-GTP undergoes When active, MLCK phosphorylates MLC permit- post-translational geranylgeranylation and migra- ting cross bridge formation and contraction. Besides tion to the cell membrane in an active form. The this Ca2+-dependent contractile mechanism, there 2+ process of activation of RhoA is regulated by three is also evidence for a Ca -independent mechanism groups of proteins: guanine nucleotide dissociation operating to maintain smooth muscle contraction. inhibitors (GDI) inhibits RhoA activation, guanine This mechanism was suggested based on the nucleotide exchange factors (GEF) which promote observation that a simple relationship between 2+ the exchange of GDP for GTP to activate RhoA and [Ca ]i and agonist-induced force generation does GTPase associated proteins (GAP) which promote not necessarily exist in vascular smooth muscle intrinsic GTPase activity to inhibit RhoA activation. cells.15 Agonist stimulation results in a transitory 2+ Theoretically, factors that disrupt RhoA activation rise in calcium but [Ca ]i falls to near prestimula- altering the activity of any of these proteins, by tion levels even in the continued presence of the agonist and force generation. These experiments 2+ demonstrate that besides increasing [Ca ]i in a transient fashion, vasoconstrictor agents may in- RHOA~GDP 2+ (INACTIVE) crease the Ca sensitivity of the contractile appa- BINDS GTP 2+ ratus. The Ca sensitization likely involves the MODIFICATIONS RhoA/Rho-kinase regulation of MLC phosphatase MIGRATES TO MEMBRANE 16 RHOA~GTP activity. (ACTIVE)

MLC + RHO-KINASE (RELAXATION) +ATP Vasorelaxation and erection of the penis MLC KINASE MLC PHOSPHATASE MLC PHOSPHATASE~P (ACTIVE) (INACTIVE)

The penis becomes erect with the relaxation of MLC ~P 7,17 PHOSPHATASE arteriolar and sinusoidal smooth muscle. The (CONTRACTION) PHOSPHATASE (?) principle agent leading to penile smooth muscle Figure 1 The RhoA/Rho-kinase pathway in the maintenance of relaxation is NO released by the autonomic innerva- smooth muscle contraction. RhoA is activated when it binds GTP, tion of the erectile tissue and by arteriolar and undergoes geranylgeranylation and migrates to the cell mem- 18 cavernosal endothelial cells. NO diffuses into brane. Activated RhoA activates Rho-kinase which catalyzes the smooth muscle cells where it activates soluble phosphorylation (and inactivation) of MLC phosphatase. When guanylate cyclase elevating intracellular levels of MLC phosphatase is in the active form (ie, nonphosphorylated), it catalyzes the dephosphorylation of MLC and thereby promotes cyclic GMP and activating cyclic GMP-dependent 19 smooth muscle relaxation. In the penile circulation, smooth protein kinase (PKG). The NO/cGMP/PKG path- muscle relaxation leads to increased intracavernosal blood way is thought to reduce contractile activity by: (1) pressure and erection.

International Journal of Impotence Research Erectile response TM Mills et al S22 preventing geranylgeranylation or blocking migra- cavernous tissue were contracted with phenylephr- tion would block smooth muscle contraction and ine or endothelin-1 and then caused to relax in a allow relaxation. Activated RhoA has a variety of dose-dependent fashion with the addition of Y- downstream targets, but in penile smooth muscle, 27632.3,4,21,22 Based on these in vivo and in vitro the target of interest is Rho-kinase. Rho-kinase studies, it can be suggested that the RhoA/Rho- catalyzes the phosphorylation of MLC phosphatase kinase pathway plays a critical role in the main- which inhibits the activity of this enzyme. When tenance of the penis in the nonerect state. Further- MLC phosphatase activity is low, MLC remains more, upregulation of this pathway could be a predominantly in the phosphorylated form and component in erectile dysfunction of several etiol- smooth muscle contraction is maintained. Interrup- ogies.1,3–5,8,21,22,25 It is of interest that strips of rabbit tion of the process by which RhoA activates Rho- which have been contracted with pheny- kinase would reverse smooth muscle contraction lephrine relax with the addition of Y-27632 suggest- leading to relaxation. ing that the RhoA/Rho-kinase pathway may be important in the sexual response and sexual dysfunction in females as well.26

RhoA/Rho-kinase activity in the circulation of the penis Use of a dominant-negative form of RhoA in the investigation of the role of the RhoA/ Reports from several laboratories have established Rho-kinase pathway in erection the presence of elements of the RhoA/Rho-kinase signaling pathway in the penile smooth muscle.1,3– Additional proof supporting the importance of the 5,21,22 These reported studies have utilized Western RhoA/Rho-kinase pathway in the erectile response analysis and immunocytochemistry to demonstrate comes from the recent studies of Chitaley et al.27 the presence of both RhoA and Rho-kinase proteins. These experiments utilized the adeno-associated In studies of the role of the RhoA/Rho-kinase viral gene transfer of a dominant-negative RhoA pathway in erection, many investigators have uti- mutant in penile tissue. The dominant-negative lized the selective Rho-kinase inhibitor, Y-27632 form of RhoA binds GTP and is modified and [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclo- migrates to the cell membrane as normal. However, hexanecarboxamide dihydrochloride, monohy- the dominant-negative RhoA fails to activate Rho- drate], Mitsubishi Pharma, Osaka, Japan).1,5,22 This kinase so that MLC phosphatase remains active to pyridine derivative is thought to occupy the ATP dephosphorylate MLCBP and smooth muscle re- binding site on Rho-kinase and thereby prevent the laxation occurs. When the dominant-negative RhoA phosphorylation of MLC phosphatase. In the non- was injected into the cavernosal sinuses of rats, phosphorylated state, MLC phosphatase is active to smooth muscle cells were infected as demonstrated remove the high-energy phosphate group from MLC by Western analysis and immunohistochemistry. In and promote smooth muscle relaxation. Injection of the infected animals, basal intracavernosal pressure Y-27632 into the cavernous sinuses of rats increases was significantly elevated and the erectile response intracavernosal pressure (ICP) in a dose-dependent to ganglionic stimulation was strongly enhanced. fashion.22 Prior treatment with inhibitors of NOS or Systemic blood pressure was unaffected by the inhibitors of guanylate cyclase fails to alter the treatment. This novel finding strongly supports the vasodilatory action of Y-27632 demonstrating that hypothesis that the RhoA/Rho-kinase pathway is the Rho-kinase inhibitor does not activate NO- critically involved in the maintenance of the penis dependent vasorelaxation to cause erection. Activa- in the nonerect state and raises the possibility that tion of the RhoA/Rho-kinase pathway appears to the use of genetic inhibitors of RhoA activation mediate the constrictor action of ET-1 and could be of value in the treatment of erectile a-adrenergic agonists. In these studies, rats received dysfunction. an intracavernosal injection of methoxamine (a-adrenergic agonist) or endothelin-1 (both are potent vasoconstrictors in the cavernous circula- tion12,23,24). However, the strong vasoconstrictor RhoA/Rho-kinase mediated action of these agents was reversed by prior vasoconstriction must be overcome for treatment with Y-27632 to inhibit RhoA/Rho-kinase erection to occur activity.1 These in vivo results suggest that the RhoA/Rho-kinase pathway mediates the vasocon- striction resulting from activation of both the a- Methoxamine and ET-1 are potent vasoconstrictors adrenergic or ET-1 pathways in the penile circula- in the cavernosal circulation.12,28 However, vaso- tion. This concept is also supported by several in constriction in response to these agents was pre- vitro studies in which strips of human, rabbit or rat vented and erection was of normal magnitude when

International Journal of Impotence Research Erectile response TM Mills et al S23 methoxamine or endothelin-1 was injected during NO donor drug plus Y-27632) was significantly electrically or NO-induced erection.23,24 This ob- greater than that of the response to either drug alone servation suggests that vasoconstrictor pathways are or to the sum of the individual responses (Figure 2). overcome during erection and raise the possibility Taken together, these various lines of investigation that the primary action of NO leading to erection is strongly support the hypothesis that NO/cGMP/ to inhibit the vasoconstriction mediated by the PKG-mediated vasodilation leading to erection RhoA/Rho-kinase pathway. involves inhibition of RhoA/Rho-kinase-regulated vasoconstriction.22

NO-dependent inhibition of RhoA/Rho-kinase-mediated vasoconstric- Conclusions tion Studies of the erectile process have demonstrated A review of the vascular smooth muscle literature that the penis is maintained in the nonerect state by revealed strong evidence for inhibition of RhoA/ activity of the RhoA/Rho-kinase pathway. The Rho-kinase signaling by the NO/cGMP/PKG path- activity of this pathway maintains the state of way in other vascular beds. Sauzeau et al19 showed contraction of arteriolar and sinusoidal smooth that NO-regulated vasodilation was due, in part, to muscle to limit blood flow and keeps the penis the PKG-dependent phosphorylation of RhoA. The flaccid. We have proposed that the principle action phosphorylation is thought to destabilize membrane of NO leading to erection is by inhibiting the RhoA/ binding of RhoA. Other studies29 showed that rat Rho-kinase signaling pathway to permit smooth aortic strips contracted in response to phenylephr- muscle. With relaxation of the arteriolar smooth ine, but relaxed when treated with Y-27632 to muscle, blood flows into the cavernous sinuses inhibit Rho-kinase activity. However, if the strips exerting shear and stretch force to stimulate eNOS were denuded of their endothelial layer (the primary activity and further relaxation. As the sinusoidal source of NO in isolated aorta), Y-27632 was less smooth muscle relaxes, blood fills the sinuses to effective at causing relaxation, but with the addition activate veno-occlusion and results in erection. of a NO donor drug to the aortic strips, the relaxation effect of Y-27632 was fully restored. In other in vivo studies, the erectile response to a short-acting NO donor drug (NOR-1) was measured before and after References administration of a threshold dose of Y-27632.30 The very low dose of Y-27632 significantly enhanced the 1 Mills TM et al. Effect of Rho-kinase inhibition on vasocon- erectile response to the NO donor drug and further, striction in the penile circulation. J Appl Physiol 2001; 91: the response to the combination of the two drugs (ie, 1269 – 1273. 2 Mills TM, Chitaley K, Lewis R. Vasoconstrictors in erectile physiology. Int J Impot Res 2001; 13: S29 – S34. 3 Rees RW et al. Human and rabbit cavernosal smooth muscle 0.8 cells express Rho-kinase. Int J Impot Res 2002; 14: 1–7. 4 Rees RW et al. Y-27632, an inhibitor of Rho-kinase, antag- onizes noradrenergic contractions in the rabbit and human penile corpus cavernosum. Br J Pharmacol 2001; 133: 455 – 0.6 458. 5 Chitaley K, Webb RC, Mills TM. RhoA/Rho-kinase: a novel player in the regulation of penile erection. Int J Impot Res 0.4 2001; 13: 67 – 72. 6 Andersson KE. Pharmacology of erectile function and dys- function. Urol Clin NA 2001; 28: 233 – 247. ICP/MAP 0.2 7 Andersson KE. Pharmacology of penile erection. Pharmacol Rev 2001; 53: 417 – 450. 8 Andersson KE. Neurophysiology/pharmacology of erection. Int J Impot Res 2001; 13: S8 – S17. 0.0 9 Naylor AM. Endogenous neurotransmitters mediating penile NOR-1 Y-27632 NOR-1 erection. Br J Urol 1998; 81: 424 – 431. 10 Redmond EM et al. Regulation of endothelin receptors by Figure 2 Effect of inhibition of the Rho-kinase signaling path- nitric oxide in cultured rat vascular smooth muscle cells. J Cell way on the erectile response (ICP/MAP) to intracavernosal Physiol 1996; 166: 469 – 479. injection of the NO-donor drug, NOR-1 (2-mg). Note that the 11 Traish A, Kim NN, Moreland RB, Goldstein I. Role of alpha NOR-1 response is strongly enhanced when given in conjunction adrenergic receptors in erectile function. Int J Impot Res 2000; with a near-threshold dose of the Rho-kinase inhibitor, Y-27632 12: S48 – S63. (o2 nmol). These results support the hypothesis that NO inhibits 12 Dai Y et al. Receptor-specific influence of endothelin-1 in the Rho-kinase-mediated vasoconstriction as part of the erectile erectile response of the rat. Am J Physiol 2000; 279: R25 – R30. response. *Indicates significant difference from NOR-1 only. 13 Pollock DM, Tasker AS. In: Pollock DM, Highsmith RF (eds). After Mills et al, Eur J Pharmacol 2002; 439: 173. Endothelin Receptors and Receptor Antagonists, Endothelin

International Journal of Impotence Research Erectile response TM Mills et al S24 Receptors and Signaling Mechanisms. Springer: Berlin, 23 Mills T et al. Endothelin-1 induced vasoconstriction is Heidelberg, New York, 1998, pp 3 – 15 inhibited during erection in rats. Am J Physiol 2001; 281: 14 Somlyo AP, Wu X, Walker LA, Somlyo AV. Pharmacomecha- R476 – R483. nical coupling: the role of calcium, G-proteins, kinases and 24 Wingard C, Lewis RW, Mills TM. Erection and NO override the phosphatases. Rev Physiol Biochem Pharmacol 1999; 134: vasoconstrictor effect of alpha adrenergic stimulation in the 201 – 234. rat penile vasculature. Int J Impot Res 2001; 13: 212 – 220. 15 DeFeo T, Morgan K. Calcium-force relationships as detected 25 Chang S et al. Increased contractility of diabetic rabbit corpora with Aequorin in two different vascular smooth muscles of the smooth muscle in response to endothelin is mediated via Rho- ferret. J Physiol 1985; 369: 269 – 292. kinase beta. Int J Impot Res 2003; 15: 53 – 62. 16 Somlyo AP, Somlyo AV. Signal transduction by G-proteins, 26 Park JK et al. Role of Rho-kinase activity in angiotensin II- rho-kinase and protein phosphatase to smooth muscle and mediated contraction of rabbit clitoral cavernosum smooth non-muscle myosin II. J Physiol 2000; 522 (Part 2): 177 – 185. muscle. Int J Impot Res 2003; 14: 472 – 477. 17 Stief CG, Noack T, Andersson KE. Signal transduction in 27 Chitaley K et al. Adeno-associated viral gene transfer of cavernous smooth muscle. World J Urol 1997; 15: 27 – 31. dominant negative RhoA enhances erectile function in rats. 18 Burnett AL. The role of nitric oxide in the physiology of Biochem Biophys Res Comm 2002; 298: 427 – 432. erection (A Minireview). Biol Reprod 1995; 52: 485 – 489. 28 Reilly CM, Stopper VS, Mills TM. Androgens modulate the 19 Sauzeau V et al. Cyclic GMP-dependent protein kinase alpha-adrenergic responsiveness of vascular smooth muscle in signaling pathway inhibits RhoA-induced Ca2+ sensitization the corpus cavernosum. J Androl 1997; 18: 26 – 31. of contraction in vascular smooth muscle. J Biol Chem 2000; 29 Chitaley K, Weber D, Webb RC. RhoA/Rho-kinase, vascular 275: 21722 – 21729. changes, and hypertension. Curr Hyperten Rep 2001; 3: 139 – 20 de Tejada IS. Molecular mechanisms for the regulation of 144. penile smooth muscle. Int J Impot Res 2002; 14: S6 – S10. 30 Mills TM, Chitaley K, Lewis RW, Webb RC. Nitric oxide 21 Wang H et al. RhoA-mediated Ca2+ sensitization in erectile inhibits RhoA/Rho-kinase signaling to cause erection. Eur J function. J Biol Chem 2003; 277: 30614 – 30621. Pharmacol 2002; 439: 173 – 174. 22 Chitaley K et al. Antagonism of Rho-kinase stimulates rat penile erection via a nitric oxide-independent pathway. Nat Med 2001; 7: 119 – 122.

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