Atorvastatin But Not Elocalcitol Increases Sildenafil Responsiveness in Spontaneously Hypertensive Rats by Regula the RhoA/ROCK Pathway BENEDETTA FIBBI,*5 ANNAMARIA MORELLI,*5 MIRCA MARINI,{ XIN-HUA ZHANG,* ROSA MANCINA,* LINDA VIGNOZZI,* SANDRA FILIPPI,{ ARAVINDA CHAVALMANE,* ENRICO SILVESTRINI,* ENRICO COLLI,§ LUCIANO ADORINI,§ GABRIELLA BARBARA VANNELLI,{ AND MARIO MAGGI* From the *Andrology Unit, Department of Physiopathology, Center for Research, Transfer and High Education DENOTHE, the Department of Anatomy, Histology and Forensic Medicine, and the `Interdepartmental Laboratory Functional and Cellular Pharmacology of Reproduction, Departments of Pharmacology and Clinical Physiopatholo University of Florence, Florence, Italy; and §Bioxell, Milan, Italy. ABSTRACT: Spontaneously hypertensive rats (SHR) are charac- terized by impaired erectile function and overactivity of the procontractile RhoA/Rho-associated, coiled-coil–containing protein kinase (RhoA/ROCK) pathway, as compared with their normotensive counterpart,Wistar-Kyoto rats.By measuring the intracavernous pressure:mean arterial pressure (ICP:MAP) ratio after electrostimu- lation of the cavernous nerve,we confirmed these findings and showed that responsiveness to sildenafil (25 mg/kg by oral gavage) also is hampered in SHR.A 2-week treatment with atorvastatin (5 and 30 mg/kg)improved the sildenafil-induced ICP:MAP increase and normalized RhoA and ROCK2 overexpression in SHR corpora cavernosa (CC). Conversely,other genes, neuronalnitric oxide synthase (NOS),endothelial NOS, and phosphodiesterase 5, were unaffected.In human fetalsmooth muscle cells derived from CC (hfPSMC), atorvastatin inhibited RhoA membrane translocation and ROCK activity, as well as RhoA-dependent biologic functions like cell migration and cell proliferation. Atorvastatin’s effect on migration was rescued in a dose-dependent manner by geranylgeranyl pyrophos- phate,suggesting the involvement of RhoA geranylgeranylation. In hfPSMC, atorvastatin decreased the expression of RhoA-dependent genes such as ROCK2, desmin, a-smooth muscle actin, SM22a, and myocardin. In contrast to atorvastatin, elocalcitol, a vitamin D analog that also interferes with RhoA activation in SHR bladder, was unable to restore penile responsiveness to sildenafil. In conclusion, atorvastatin, but not elocalcitol, ameliorates sildenafil-induced penile erections in SHR, likely by interfering with RhoA/ROCK signaling within the penis. Key words: Erectile dysfunction, phosphodiesterase 5 inhibitor, human penile smooth muscle cells, RhoA-dependent genes,SHR model. J Androl 2008;29:70–84 S everalcontractile systems are responsible for main- taining the penis in the contracted/flaccid state, its predominant physiologic condition. The mostcharac- terized ones are endothelin 1 (ET1) and noradrenaline (NA), which mediate their effects via a receptor-coupled intracellular calcium rise (Maggi et al,2000;Morelli et al, 2005).Although this receptor-mediated intracellular calcium increase is transient, penile smooth muscle (SM) cells are able to maintain the contracted state thereafter via a calcium-sensitizing pathway. This pathway consists of the activation of parallel pathways able to increase calcium sensitivity even at the same intracellular calcium levels (Morelliet al,2005),and a primary mechanism involves the RhoA/Rho kinase pathway (Wang et al, 2002). Accordingly, the selective inhibitor of Rho kinase activity, Y-27632, has been shown to cause relaxation of human corpora cavernosa (CC) in vitro and to induce penile erection in animal models(Mills et al, 2001). Moreover,transfection of a RhoA dominantnegative mutant was found to enhance erectile function in rats (Bivalacqua etal, 2004).Hence,prevention ofRhoA activation could ameliorate penileerection,whereas enhancement of RhoA activity appearsto mediate erectile dysfunction (ED). RhoA is a member ofa small monomeric GTPase family that is involved in SM contraction and the regulation ofseveralother SM-dependentprocesses such as cell adhesion (Ren et al, 1999), motility (Wheeler 5 These authors contributed equally to the article. This study wassupported by a grantfrom Progettidi Ricerca Scientifica di Rilevante Interesse Nazionale (PRIN2005-MIUR) and by a scientific grant from Pfizer Italia (Rome, Italy). Correspondence to: Dr Mario Maggi, Andrology Unit, Department of Clinical Physiopathology, University of Florence, V.le G. Pieraccini, 6, 50139 Florence, Italy (e-mail: m.maggi@dfc.unifi.it). Received for publication April 23,2007;accepted for publication August 9, 2007. DOI: 10.2164/jandrol.107.003152 Journal of Andrology, Vol. 29, No. 1, January/February 2008 Copyright E American Society of Andrology 70