Advertisement

Sildenafil Promotes eNOS Activation and Inhibits NADPH Oxidase in the Transgenic Sickle Cell Mouse Penis

      Abstract

      Introduction

      Sickle cell disease (SCD)‐associated vasculopathy in the penis is characterized by aberrant nitric oxide and phosphodiesterase (PDE) 5 signaling, and by increased oxidative stress. Preliminary clinical trials show that continuous treatment with PDE5 inhibitor sildenafil unassociated with sexual activity decreases priapic activity in patients with SCD. However, the mechanism of its vasculoprotective effect in the penis remains unclear.

      Aims

      We evaluated whether continuous administration of PDE5 inhibitor sildenafil promotes eNOS function at posttranslational levels and decreases superoxide‐producing enzyme NADPH oxidase activity in the sickle cell mouse penis.

      Methods

      SCD transgenic mice were used as an animal model of SCD. WT mice served as controls. Mice received treatment with the PDE5 inhibitor sildenafil (100 mg/kg/day) or vehicle for 3 weeks. eNOS phosphorylation on Ser‐1177 (positive regulatory site), eNOS interactions with heat‐shock protein 90 (HSP90) (positive regulator), phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser‐1177), an NADPH oxidase catalytic subunit gp91(phox), and a marker of oxidative stress (4‐hydroxy‐2‐nonenal [HNE]) were measured by Western blot.

      Main Outcome Measures

      Effect of continuous sildenafil treatment on eNOS posttranslational activation, NADPH oxidase catalytic subunit, and oxidative stress in the penis of the sickle cell mouse.

      Results

      Continuous treatment with sildenafil reversed (P < 0.05) the abnormalities in protein expressions of P‐eNOS (Ser‐1177), eNOS/HSP90 interaction, P‐AKT, protein expression of gp91(phox), and 4‐HNE, in the sickle cell mouse penis. Sildenafil treatment of WT mice did not affect any of these parameters.

      Conclusion

      Our findings that sildenafil enhances eNOS activation and inhibits NADPH oxidase function in the sickle cell mouse penis offers a vasculoprotective molecular basis for the therapeutic effect of sildenafil in the penis in association with SCD. Musicki B, Bivalacqua TJ, Champion HC, and Burnett AL. Sildenafil promotes eNOS activation and inhibits NADPH oxidase in the transgenic sickle cell mouse penis. J Sex Med 2014;11:424–430.

      Key Words

      To read this article in full you will need to make a payment
      ISSM Member Login
      Login with your ISSM username and password.
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Purchase one-time access:

      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Kato G.J.
        • Hebbel R.P.
        • Steinberg M.H.
        • Gladwin M.T.
        Vasculopathy in sickle cell disease: Biology, pathophysiology, genetics, translational medicine, and new research directions.
        Am J Hematol. 2009; 84: 618-625
        • Wood K.C.
        • Hsu L.L.
        • Gladwin M.T.
        Sickle cell disease vasculopathy: A state of nitric oxide resistance.
        Free Radic Biol Med. 2008; 44: 1506-1528
        • Hoppe C.C.
        Novel therapies targeting the endothelium in sickle cell disease.
        Hemoglobin. 2011; 35: 530-546
        • Hsu L.L.
        • Champion H.C.
        • Campbell‐Lee S.A.
        • Bivalacqua T.J.
        • Manci E.A.
        • Diwan B.A.
        • Schimel D.M.
        • Cochard A.E.
        • Wang X.
        • Schechter A.N.
        • Noguchi C.T.
        • Gladwin M.T.
        Hemolysis in sickle cell mice causes pulmonary hypertension due to global impairment in nitric oxide bioavailability.
        Blood. 2007; 109: 3088-3098
        • Champion H.C.
        • Bivalacqua T.J.
        • Takimoto E.
        • Kass D.A.
        • Burnett A.L.
        Phosphodiesterase‐5A dysregulation in penile erectile tissue is a mechanism of priapism.
        Proc Natl Acad Sci U S A. 2005; 102: 1661-1666
        • Kato G.J.
        • Taylor 6th., J.G.
        Pleiotropic effects of intravascular haemolysis on vascular homeostasis.
        Br J Haematol. 2010; 148: 690-701
        • Pritchard Jr., K.A.
        • Ou J.
        • Ou Z.
        • Shi Y.
        • Franciosi J.P.
        • Signorino P.
        • Kaul S.
        • Ackland‐Berglund C.
        • Witte K.
        • Holzhauer S.
        • Mohandas N.
        • Guice K.S.
        • Oldham K.T.
        • Hillery C.A.
        Hypoxia‐induced acute lung injury in murine models of sickle cell disease.
        Am J Physiol Lung Cell Mol Physiol. 2004; 286: L705-714
        • Wood K.C.
        • Hebbel R.P.
        • Granger D.N.
        Endothelial cell NADPH oxidase mediates the cerebral microvascular dysfunction in sickle cell transgenic mice.
        FASEB J. 2005; 19: 989-991
        • Wood K.C.
        • Hebbel R.P.
        • Lefer D.J.
        • Granger D.N.
        Critical role of endothelial cell‐derived nitric oxide synthase in sickle cell disease‐induced microvascular dysfunction.
        Free Radic Biol Med. 2006; 40: 1443-1453
        • Broderick G.A.
        Priapism and sickle‐cell anemia: Diagnosis and nonsurgical therapy.
        J Sex Med. 2012; 9: 88-103
        • Burnett A.L.
        • Bivalacqua T.J.
        • Champion H.C.
        • Musicki B.
        Long‐term oral phosphodiesterase 5 inhibitor therapy alleviates recurrent priapism.
        Urology. 2006; 67: 1043-1048
        • Burnett A.L.
        • Bivalacqua T.J.
        • Champion H.C.
        • Musicki B.
        Feasibility of the use of phosphodiesterase type 5 inhibitors in a pharmacologic prevention program for recurrent priapism.
        J Sex Med. 2006; 3: 1077-1084
        • Burnett A.L.
        • Bivalacqua T.J.
        Priapism: Current principles and practice.
        Urol Clin North Am. 2007; 34: 631-642
        • Musicki B.
        • Champion H.C.
        • Hsu L.L.
        • Bivalacqua T.J.
        • Burnett A.L.
        Posttranslational inactivation of endothelial nitric oxide synthase in the transgenic sickle cell mouse penis.
        J Sex Med. 2011; 8: 419-426
        • Lagoda G.
        • Sezen S.F.
        • Cabrini M.R.
        • Musicki B.
        • Burnett A.L.
        Molecular analysis of erection regulatory factors in sickle cell disease associated priapism in the human penis.
        J Urol. 2013; 189: 762-768
        • Bivalacqua T.J.
        • Musicki B.
        • Hsu L.L.
        • Berkowitz D.E.
        • Champion H.C.
        • Burnett A.L.
        Sildenafil citrate‐restored eNOS and PDE5 regulation in sickle cell mouse penis prevents priapism via control of oxidative/nitrosative stress.
        PLoS ONE. 2013; 8: e68028
        • Kanika N.D.
        • Melman A.
        • Davies K.P.
        Experimental priapism is associated with increased oxidative stress and activation of protein degradation pathways in corporal tissue.
        Int J Impot Res. 2010; 22: 363-373
        • Musicki B.
        • Liu T.
        • Sezen S.F.
        • Burnett A.L.
        Targeting NADPH oxidase decreases oxidative stress in the transgenic sickle cell mouse penis.
        J Sex Med. 2012; 9: 1980-1987
        • Bivalacqua T.J.
        • Musicki B.
        • Hsu L.L.
        • Gladwin M.T.
        • Burnett A.L.
        • Champion H.C.
        Establishment of a transgenic sickle‐cell mouse model to study the pathophysiology of priapism.
        J Sex Med. 2009; 6: 2494-2504
        • Claudino M.A.
        • Franco‐Penteado C.F.
        • Corat M.A.
        • Gimenes A.P.
        • Passos L.A.
        • Antunes E.
        • Costa F.F.
        Increased cavernosal relaxations in sickle cell mice priapism are associated with alterations in the NO‐cGMP signaling pathway.
        J Sex Med. 2009; 6: 2187-2196
        • Bivalacqua T.J.
        • Ross A.E.
        • Strong T.D.
        • Gebska M.A.
        • Musicki B.
        • Champion H.C.
        • Burnett A.L.
        Attenuated RhoA/Rho‐kinase signaling in penis of transgenic sickle cell mice.
        Urology. 2010; 76: 510.e7-510.e12
        • Mi T.
        • Abbasi S.
        • Zhang H.
        • Uray K.
        • Chunn J.L.
        • Xia L.W.
        • Molina J.G.
        • Weisbrodt N.W.
        • Kellems R.E.
        • Blackburn M.R.
        • Xia Y.
        Excess adenosine in murine penile erectile tissues contributes to priapism via A2B adenosine receptor signaling.
        J Clin Invest. 2008; 118: 1491-1501
        • Wen J.
        • Jiang X.
        • Dai Y.
        • Zhang Y.
        • Tang Y.
        • Sun H.
        • Mi T.
        • Phatarpekar P.V.
        • Kellems R.E.
        • Blackburn M.R.
        • Xia Y.
        Increased adenosine contributes to penile fibrosis, a dangerous feature of priapism, via A2B adenosine receptor signaling.
        FASEB J. 2010; 24: 740-749
        • Kanika N.D.
        • Tar M.
        • Tong Y.
        • Kuppam D.S.
        • Melman A.
        • Davies K.P.
        The mechanism of opiorphin‐induced experimental priapism in rats involves activation of the polyamine synthetic pathway.
        Am J Physiol Cell Physiol. 2009; 297: C916-927
        • Pászty C.
        • Brion C.M.
        • Manci E.
        • Witkowska H.E.
        • Stevens M.E.
        • Mohandas N.
        • Rubin E.M.
        Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease.
        Science. 1997; 278: 876-878
        • Bivalacqua T.J.
        • Sussan T.E.
        • Gebska M.A.
        • Strong T.D.
        • Berkowitz D.E.
        • Biswal S.
        • Burnett A.L.
        • Champion H.C.
        Sildenafil inhibits superoxide formation and prevents endothelial dysfunction in a mouse model of secondhand smoke induced erectile dysfunction.
        J Urol. 2009; 181: 899-906
        • Hurt K.J.
        • Musicki B.
        • Palese M.A.
        • Crone J.C.
        • Becker R.E.
        • Moriaruty J.L.
        • Snyder S.H.
        • Burnett A.L.
        Akt‐dependent phosphorylation of endothelial nitric oxide synthase mediated penile erection.
        Proc Natl Acad Sci U S A. 2002; 99: 4061-4066
        • Milano G.
        • Bianciardi P.
        • Rochemont V.
        • Vassalli G.
        • Segesser L.K.
        • Corno A.F.
        • Guazzi M.
        • Samaja M.
        Phosphodiesterase‐5 inhibition mimics intermittent reoxygenation and improves cardioprotection in the hypoxic myocardium.
        PLoS ONE. 2011; 6: e27910
        • Musicki B.
        • Champion H.C.
        • Becker R.E.
        • Liu T.
        • Kramer M.F.
        • Burnett A.L.
        Erection capability is potentiated by long‐term sildenafil treatment: Role of blood flow‐induced endothelial nitric‐oxide synthase phosphorylation.
        Mol Pharmacol. 2005; 68: 226-232
        • Fleming I.
        Molecular mechanisms underlying the activation of eNOS.
        Pflugers Arch. 2010; 459: 793-806
        • Herranz B.
        • Marquez S.
        • Guijarro B.
        • Aracil E.
        • Aicart‐Ramos C.
        • Rodriguez‐Crespo I.
        • Serrano I.
        • Rodríguez‐Puyol M.
        • Zaragoza C.
        • Saura M.
        Integrin‐linked kinase regulates vasomotor function by preventing endothelial nitric oxide synthase uncoupling: Role in atherosclerosis.
        Circ Res. 2012; 110: 439-449
        • Boo Y.C.
        • Jo H.
        Flow‐dependent regulation of endothelial nitric oxide synthase: Role of protein kinases.
        Am J Physiol Cell Physiol. 2003; 285: C499-508
        • Mondaini N.
        • Ponchietti R.
        • Muir G.H.
        • Montorsi F.
        • Di Loro F.
        • Lombardi G.
        • Rizzo M.
        Sildenafil does not improve sexual function in men without erectile dysfunction but does reduce the postorgasmic refractory time.
        Int J Impot Res. 2003; 15: 225-228
        • Behr‐Roussel D.
        • Oudot A.
        • Caisey S.
        • Coz O.L.
        • Gorny D.
        • Bernabé J.
        • Wayman C.
        • Alexandre L.
        • Giuliano F.A.
        Daily treatment with sildenafil reverses endothelial dysfunction and oxidative stress in an animal model of insulin resistance.
        Eur Urol. 2008; 53: 1272-1280
        • Behr‐Roussel D.
        • Oudot A.
        • Compagnie S.
        • Gorny D.
        • Le Coz O.
        • Bernabe J.
        • Wayman C.
        • Alexandre L.
        • Giuliano F.
        Impact of a long‐term sildenafil treatment on pressor response in conscious rats with insulin resistance and hypertriglyceridemia.
        Am J Hypertens. 2008; 21: 1258-1263
        • Oudot A.
        • Behr‐Roussel D.
        • Le Coz O.
        • Poirier S.
        • Bernabe J.
        • Alexandre L.
        • Giuliano F.
        How does chronic sildenafil prevent vascular oxidative stress in insulin‐resistant rats?.
        J Sex Med. 2010; 7: 79-88
        • Ebrahimi F.
        • Shafaroodi H.
        • Asadi S.
        • Nezami B.G.
        • Ghasemi M.
        • Rahimpour S.
        • Hashemi M.
        • Doostar Y.
        • Dehpour A.R.
        Sildenafil decreased cardiac cell apoptosis in diabetic mice: Reduction of oxidative stress as a possible mechanism.
        Can J Physiol Pharmacol. 2009; 87: 556-564
        • Yaguas K.
        • Bautista R.
        • Quiroz Y.
        • Ferrebuz A.
        • Pons H.
        • Franco M.
        • Vaziri N.D.
        • Rodriguez‐Iturbe B.
        Chronic sildenafil treatment corrects endothelial dysfunction and improves hypertension.
        Am J Nephrol. 2010; 31: 283-291
        • Schafer A.
        • Fraccarollo D.
        • Pfortsch S.
        • Flierl U.
        • Vogt C.
        • Pfrang J.
        • Kobsar A.
        • Renné A.
        • Eigenthaler M.
        • Ertl G.
        • Bauersachs J.
        Improvement of vascular function by acute and chronic treatment with the PDE‐5 inhibitor sildenafil in experimental diabetes mellitus.
        Br J Pharmacol. 2008; 153: 886-893
        • Luo L.
        • Dai D.Z.
        • Cheng Y.S.
        • Zhang Q.
        • Yuan W.J.
        • Dai Y.
        Sildenafil improves diabetic vascular activity through suppressing endothelin receptor A, iNOS and NADPH oxidase which is comparable with the endothelin receptor antagonist CPU0213 in STZ‐injected rats.
        J Pharm Pharmacol. 2011; 63: 943-951
        • Helmy M.M.
        • Senbel A.M.
        Evaluation of vitamin E in the treatment of erectile dysfunction in aged rats.
        Life Sci. 2012; 90: 489-494
        • Mostafa T.
        • Rashed L.
        • Kotb K.
        • Taymour M.
        Effect of testosterone and frequent low‐dose sildenafil/tadalafil on cavernous tissue oxidative stress of aged diabetic rats.
        Andrologia. 2012; 44: 411-415
        • Selemidis S.
        • Dusting G.J.
        • Peshavariya H.
        • Kemp‐Harper B.K.
        • Drummond G.R.
        Nitric oxide suppresses NADPH oxidase‐dependent superoxide production by S‐nitrosylation in human endothelial cells.
        Cardiovasc Res. 2007; 75: 349-358
        • Qian J.
        • Chen F.
        • Kovalenkov Y.
        • Pandey D.
        • Moseley M.A.
        • Foster M.W.
        • Black S.M.
        • Venema R.C.
        • Stepp D.W.
        • Fulton D.J.
        Nitric oxide reduces NADPH oxidase 5 (Nox5) activity by reversible S‐nitrosylation.
        Free Radic Biol Med. 2012; 52: 1806-1819
        • Duerrschmidt N.
        • Stielow C.
        • Muller G.
        • Pagano P.J.
        • Morawietz H.
        NO‐mediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells.
        J Physiol. 2006; 576: 557-567