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 erection. 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 penis 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 tumescence events (non- sexual erections 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