Phosphodiesterase-9 (PDE9) Inhibition with BAY 73-6691 Increases Corpus Cavernosum Relaxations Mediated by Nitric Oxide–Cyclic GMP Pathway in Mice

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ORIGINAL ARTICLE Phosphodiesterase-9 (PDE9) inhibition with BAY 73-6691 increases corpus cavernosum relaxations mediated by nitric oxide–cyclic GMP pathway in mice

FH da Silva1, MN Pereira1, CF Franco-Penteado2, G De Nucci1, E Antunes1 and MA Claudino3

Phosphodiesterase-9 (PDE9) specifically hydrolyzes cyclic GMP, and was detected in human corpus cavernosum. However, no previous studies explored the selective PDE9 inhibition with BAY 73-6691 in corpus cavernosum relaxations. Therefore, this study aimed to characterize the PDE9 mRNA expression in mice corpus cavernosum, and investigate the effects of BAY 73-6691 in endothelium-dependent and -independent relaxations, along with the nitrergic corpus cavernosum relaxations. Male mice received daily gavage of BAY 73-6691 (or dimethylsulfoxide) at 3 mg kg À 1 per day for 21 days. Relaxant responses to acetylcholine (ACh), nitric oxide (NO) (as acidified sodium nitrite; NaNO2 solution), sildenafil and electrical-field stimulation (EFS) were obtained in corpus cavernosum in control and BAY 73-6691-treated mice. BAY 73-6691 was also added in vitro 30 min before construction of concentration–responses and frequency curves. PDE9A and PDE5 mRNA expression was detected in the mice corpus cavernosum in a similar manner. In vitro addition of BAY 73-6691 neither itself relaxed mice corpus cavernosum nor changed the NaNO2, sildenafil and EFS-induced relaxations. However, in mice treated chronically with BAY 73-6691, the potency (pEC50) values for ACh, NaNO2 and sildenafil were significantly greater compared with control group. The maximal responses (Emax) to NaNO2 and sildenafil were also significantly greater in BAY 73-6691-treated mice. BAY 73-6691 treatment also significantly increased the magnitude and duration of the nitrergic corpus cavernosum relaxations (8–32 Hz). In conclusion, murine corpus cavernosum expresses PDE9 mRNA. Prolonged PDE9 inhibition with BAY 73-6691 amplifies the NO-cGMP-mediated cavernosal responses, and may be of therapeutic value for erectile dysfunction.

International Journal of Impotence Research (2012) 25, 69–73; doi:10.1038/ijir.2012.35; published online 4 October 2012 Keywords: cyclic GMP; erectile dysfunction; nitrergic relaxations; phosphodiesterase-5

INTRODUCTION released from nitrergic nerves and endothelial cells.10 NO Phosphodiesterases (PDEs) are a large family of enzymes that activates soluble guanylate cyclase enzyme in cavernosal specifically hydrolyze the second messengers cyclic AMP smooth muscle, resulting in accumulation of intracellular cGMP and/or cyclic GMP. There are 11 distinct PDE isoenzymes, and subsequent activation of cGMP-dependent protein kinase differing in their substrate specificity, selective inhibition or (PKG), which leads to relaxation of corpus cavernosum smooth 11 stimulation by cofactors and/or standard drugs, as well as gene muscle and penile erection. homology. The isoforms PDE5, PDE6 and PDE9 are specific for It is well established that PDE5 is the predominant isoform cGMP while PDE4, PDE7 and PDE8 are cAMP specific. The other responsible for the metabolism of cGMP in the corpus caverno- 12,13 PDEs (PDE1, PDE2, PDE3, PDE10 and PDE11) hydrolyze both cAMP sum, ceasing the erectile responses. Therefore, cGMP and cGMP.1,2 concentration in corpus cavernosum smooth muscle is mainly PDE9 expression has been detected in various tissues, including dependent on the balance between the production by soluble brain, kidney, lung, heart, small intestine, prostate, bladder, guanylate cyclase of cGMP and the breakdown by PDEs, which hematopoietic cells and corpus cavernosum.3–6 The compound represents the unique degradation pathway for this second BAY 73-6691 has been described as a potent and selective cell messenger.14 Although PDE9 is expressed in human corpus permeable PDE9 inhibitor.7 This inhibitor may have potential for cavernosum,3 no previous study investigated the effects of PDE9 treatment of neurodegenerative disorders such as Alzheimer’s inhibition in corpus cavernosum relaxations. We hypothesized that disease, improving memory via the nitric oxide (NO)/cGMP selective PDE9 inhibition with BAY 73-6691 increases the corpus pathway in rats.8,9 Moreover, BAY 73-6691 reduces the cavernosum relaxations mediated by the activation of NO-cGMP neutrophil adhesion in sickle cell disease, suggesting that PDE9 signaling pathway. Therefore, the present study aimed to inhibition in such cells may constitute an important strategy to characterize the PDE9 expression in mice corpus cavernosum, inhibit the vaso-occlusive process in this disorder.5 and investigate the endothelium-dependent and -independent Penile erection is the final result of corpus cavernosum smooth relaxations, as well as in the neurogenic nitrergic corpus muscle relaxation. This process is primarily initiated by NO cavernosum relaxations in mice pretreated with BAY 73-6691.

1Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP) Campinas, Saõ Paulo, Brazil; 2Hematology and Hemotherapy Center, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, Saõ Paulo, Brazil and 3Laboratory of Multidisciplinary Research, Saõ Francisco University (USF), Bragança Paulista, Saõ Paulo, Brazil. Correspondence: Dr MA Claudino, Laboratory of Multidisciplinary Research, Saõ Francisco University, Bragança Paulista Saõ Paulo 12916-900, Brazil. E-mail: [email protected] or [email protected] Received 23 August 2011; revised 11 June 2012; accepted 28 August 2012; published online 4 October 2012 BAY 73-6691 increases corpus cavernosum relaxation FH da Silva et al 70 MATERIALS AND METHODS 12.5 ml SYBR Green Master Mix PCR (Applied Biosystems) and 300 nM of Animals primers in a MicroAmp Optical 96-well reaction plate (Applied Biosystems) using the 7200 Sequence Detection System (Applied Biosystems). To The experimental protocols were approved by the Ethical Principles in confirm accuracy and reproducibility of real-time PCR, the intra-assay Animal Research adopted by the Brazilian College for Animal Experimenta- [ À 1/slope] tion (COBEA). Male C57BL/6 mice (30 g) were provided by Central animal precision was calculated according to the equation: E ¼ 10 , where House Services (CEMIB) of University of Campinas (UNICAMP, Saõ Paulo). the PCR efficiency was calculated. The transcripts assayed demonstrated The animals were housed five per cage on 12 h light-dark cycle, and fed a high real-time PCR efficiency rates of over 99% for primer utilized (Pearson standard chow diet with water ad libitum. correlation coefficient r40.95). Threshold cycle (Ct) was defined as the point at which the fluorescence rises appreciably above the background fluorescence. The dissociation protocol was performed at the end of each Functional studies in cavernosal strips run to check for non-specific amplification. All samples were amplified in The penis was removed and immediately placed in chilled Krebs solution duplicate, and the mean of the threshold cycle was used for further of the following composition: NaCl, 118 mM; NaHCO3, 25 mM; glucose, calculations. The probe signal was normalized to an internal reference, and 5.6 mM; KCl, 4.7 mM; KH2PO4, 1.2 mM; MgSO4 7H2O, 1.17 mM and CaCl2 GAPDH was used as reference gene and fold increase was calculated by 2H2O, 2.5 mM. After removal of the vein and urethra, the penile tissue was the conventional DDCt method. cleaned from the connective and adventitial tissue, and the fibrous septum separating the corpora cavernosa was opened from its proximal extremity Drugs and chemicals

toward the penile shaft. A slit was made in the tunica albuginea along the ACh, atropine, guanethidine, PE, NaNO2, DMSO and BAY 73-6691 were shaft to obtain two strips (B10 Â 1 Â 1 mm) of corpus cavernosum from purchased from Sigma-Aldrich (St Louis, MO, USA). Sildenafil citrate was each animal. Each strip was mounted under resting tension of 2.5 mN in obtained from Pfizer, Inc. (New York, NY, USA). All reagents used were of 10-ml organ chambers containing Krebs solution at 37 1C (pH 7.4) and analytical grade. Stock solutions were prepared in deionized water or 15 continuously bubbled with a mixture of 95% O2 and 5% CO2. Isometric DMSO and stored in aliquots at À 20 1C; dilutions were prepared force was recorded using a PowerLab 400 data acquisition system immediately before use. The responses to NO were obtained by adding (Software Chart, version 7.0; AD Instrument, Milford, MA, USA). The NaNO2 solution adjusted to pH 2.0, and concentrations of the solution in tissues were allowed to equilibrate for 1 h before starting the experiments. the bathing medium were expressed as those of NO.16–17 Cumulative concentration–response curves to ACh (1 nM to 3 mM, endothelium-dependent vasodilator), NO (added as acidified sodium nitrite Statistical analysis (NaNO2) solution; 1 nM–100 mM) or sildenafil (1 nM–10 mM) were obtained in cavernosal strips contracted with phenylephrine (PE; 10 mM, a1-adrener- Data are expressed as means±s.e.m. of n experiments. The program Instat gic receptor agonist). Non-linear regression analysis to determine the (GraphPad software) was used for statistical analysis. Analysis of variance potency (pEC50) was carried out using GraphPad Prism (GraphPad was performed, and Tukey method was chosen as a post-test. Student’s Software, San Diego, CA, USA). t-test was performed when appropriate. Po0.05 was considered stati- stically significant. Electrical-field stimulation EFS was applied in cavernosal strips placed between two platinum ring RESULTS electrodes connected to a grass S88 stimulator (Astro-Med Industrial Park, Expression of mRNA of PDE9A and PDE5 in mice corpus West Warwick, RI, USA). EFS was conducted at 50 V, 1-ms pulse width and cavernosum trains of stimuli lasting 10 s at varying frequencies (1–32 Hz). To study the PDE9A and PDE5 mRNA expression was detected in the corpus nitrergic relaxations, cavernosal tissues were pretreated with guanethidine cavernosum of mice. Both isoforms were expressed at similar (30 mM) and atropine (1 mM) to deplete the catecholamine stores and to block the cholinergic receptors, respectively. levels (Figure 1).

Concentration-dependent relaxations to ACh, NaNO2 and Experimental groups sildenafil in BAY 73-6691-treated mice Animals were divided into control group (mice received tap water alone Mice received BAY 73-6691 at 3 mg kg À 1 per day as daily oral together with 100% dimethylsulfoxide (DMSO) (200 ml) as daily gavage for 21 days) and BAY 73-6691 group (mice received BAY 73-6691 at 3 mg kg À 1 gavage for 21 days (or vehicle), after which concentration– per day as daily oral gavage for 21 days). This dose of BAY 73-6691 was response relaxing curves to ACh, NaNO2 and sildenafil were chosen according to a previous study.8 Concentration–response curves to obtained in corpus cavernosum. PE (10 mM) caused a submaximal ACh, NaNO2 and sildenafil, along with the nitrergic relaxations, were contraction in mice corpus cavernosum preparations and gener- obtained in corpus cavernosum, as detailed above. In separate experi- ated active force of 0.26±0.05 mN (n ¼ 25) from control group ments, BAY 73-6691 was added in vitro (1 mM) to the organ bath 30 min and 0.31±0.04 mN (n ¼ 21) from control BAY 73-6691 chronic before construction of concentration–responses and frequency curves. group (70–80% of the maximal response). There were no significant differences in the levels of contraction at the chosen Reverse transcription of mRNA and amplification and quantification of PDE type-5 and -9A gene expression by real-time PCR Total RNA was extracted with Trizol Reagent (Invitrogen Corp., Carlsbad, CA, USA) from corpus cavernosum mice samples. Three microgram RNA samples were incubated with 1 U DNaseI (Invitrogen, Rockville, MD, USA) for 15 min at room temperature and EDTA was added to a final concentration of 2 mM to stop the reaction. The DNaseI enzyme was subsequently inactivated by incubation at 65 1C for 5 min. DNaseI-treated RNA samples were then reverse transcribed with Superscript III and RNaseOut (Invitrogen Corp., Carlsbad, CA, USA) for 50 min at 50 1C, 15 min at 70 1C. cDNA samples were quantified using a Nanodrop spectro- photometer (ND-1000; Nanodrop Technologies Inc., Wilmington, DE, USA). Synthetic oligonucleotide primers were designed to amplify cDNA for PDE type-5 and -9A (PDE5 and PDE9A, respectively; PrimerExpress; Applied Biosystems, Foster City, CA, USA). Primer sequences for PDE5 were the Figure 1. Corpus cavernosum mRNA expressions for phosphodies- following: 50-GGAAATGGTGGGACCTTCACT-30 and reverse 50-AAGAACAA- terase-5 (PDE5) and phosphodiesterase-9A (PDE9A). The mRNA TACCACAGAATGCCA-30, primer sequences for PDE9A were the following: expression level of each gene was normalized to GAPDH expression 50-TTTAACCACAGACGACGCCA-30 and reverse 50-GTAGGGAGTGCGCTCTGA levels. Values are expressed in arbitrary units. Data represent the ATT-30. All samples were assayed in a 25-ml volume containing 10 ng cDNA, mean±s.e.m. for five mice each group.

International Journal of Impotence Research (2012), 69 – 73 & 2012 Macmillan Publishers Limited BAY 73-6691 increases corpus cavernosum relaxation FH da Silva et al 71 PE concentration. In PE-precontracted cavernosal strips, cumulative addition of ACh (1 nM–3 mM), NaNO2 (1 nM–100 mM) or sildenafil (1 nM–10 mM) produced concentration-dependent relaxations (Figures 2a–c). The potency (pEC50) values for ACh, NaNO2 and sildenafil were 2.2-, 4.7- and 5.3-fold greater (Po0.05) in cavernosal strips taken from BAY 73-6691-treated mice, respectively, compared with control group (Table 1). The maximal responses (Emax) to NaNO2 and sildenafil were also significantly greater in BAY 73-6691-treated mice (Po0.05) whereas Emax to ACh remained unchanged (Table 1). In BAY 73-6691-treated mice, sildenafil produced corpus cavernosum relaxations below the calibrated baseline (Figure 2c); therefore, in this case in particular, we recognize that caution should be taken when interpreting the pEC50 values (Table 1).

Nitrergic relaxations in BAY 73-6691-treated mice EFS (1–32 Hz) produced frequency-dependent corpus cavernosum relaxations in both control and BAY 73-6691 groups (Figure 3a). Treatment with BAY 73-6691 signiﬁcantly increased the relaxations of corpus cavernosum at frequencies of 8–32 Hz in comparison with control group. The duration of nitrergic relaxations was also signiﬁcantly increased by BAY 73-6691 treatment (Figure 3b).

Corpus cavernosum relaxations to BAY 73-6691 in vitro Addition of BAY 73-6691 (1 mM) to PE-contracted tissues did not produce mice corpus cavernosum relaxations compared with vehicle DMSO (0.2%) (Emax:20±3% and 22±2% relaxation for BAY 73-6691 and vehicle, respectively; n ¼ 5). In vitro addition of BAY 73-6691 (1 mM, 30 min) also failed to signiﬁcantly affect the NaNO2 and sildenaﬁl-induced relaxations at the level of pEC50 and Emax (Table 1). The nitrergic relaxations were also unaffected by BAY 73-6691 in vitro (8 Hz: 49±4.6% and 56±6%; 16 Hz: 50±4% and 55±5% for control and BAY 73-6691, respectively; n ¼ 5).

DISCUSSION Our present findings show that PDE9A mRNA is abundantly expressed in the mice corpus cavernosum and that the prolonged Figure 2. Concentration–response relaxing curves to acetylcholine PDE9 inhibition with BAY 73-6691 improves endothelium-dependent (ACh) (a), sodium nitrite (NaNO2)(b) and sildenafil (c) in corpus cavernosum of mice treated with vehicle or BAY 73-6691 (3 mg kg À 1 and -independent as well as nitrergic cavernosal relaxations in vitro. per day for 21 days). Data were calculated relative to the maximal The PDE9 isoform specifically hydrolyze the second messenger changes from the contraction produced by phenylephrine (PE) cGMP2,18 and therefore may constitute an important pharmacolo- (10 mM) in each tissue, which was taken as 100%. Data represent the gical target for the treatment of erectile dysfunction. Our data means±s.e.m. for 5–12 mice in each group. *Po0.05 compared with showed that PDE9A and PDE5 mRNA are expressed at similar levels the respectively control. in mice corpus cavernosum. Stimulation of non-noradrenergic,

Table 1. Effect of BAY 73-6691 in the potency (pEC50) and maximal responses (Emax) obtained from concentration–response curves to acetylcholine (ACh), NaNO2 and sildenaﬁl in mice cavernosal strips

pEC50 Emax (%)

ACh NaNO2 Sildenaﬁl ACh NaNO2 Sildenaﬁl

Control (vehicle) 7.04±0.03 6.24±0.10 6.72±0.06 60±264±693±5 BAY 73-6691 (21 days) 7.39±0.06*** 6.88±0.09*** 7.45±0.07*** 57±484±6* 117±7** BAY 73-6691 (in vitro) ND 6.02±0.06 7.19±0.03 ND 64±3.6 100±2

Abbreviations: DMSO, dimethylsulfoxide; NaNO2, sodium nitrite; ND, not determined. BAY 73-6691 (10 mM) was added in vitro to the organ bath before construction of concentration–response curves. A separate group of mice received daily oral gavage of BAY 73-6691 (3 mg kg À 1 per day for 21 days) or its vehicle (DMSO), after which concentration–response curves ACh, NaNO2 and sildenaﬁl were obtained. pEC50 is represented as À log of the molar concentration to produce 50% of the maximal contractile response elicited by ACh, NaNO2 and sildenaﬁl. Data represent mean±s.e.m. ***Po0.001; **Po0.01; *Po0.05 compared with respective control value.

& 2012 Macmillan Publishers Limited International Journal of Impotence Research (2012), 69 – 73 BAY 73-6691 increases corpus cavernosum relaxation FH da Silva et al 72

Figure 3. Magnitude (a) and duration (b) of nitrergic corpus cavernosum relaxations in corpus cavernosum of mice treated with vehicle or BAY 73-6691 (3 mg kg À 1 per day for 21 days). The duration (seconds) of the nitrergic relaxations was determined as the time elapsed from 50% relaxation to 50% recovery. Representative trace of a frequency–response curve to electrical-ﬁeld stimulation (EFS) in corpus cavernosum of mice treated with vehicle (c) or BAY 73-6691 (3 mg kg À 1 per day for 21 days) (d) contracted by phenylephrine (PE; 10 mM). Data represent the mean±s.e.m. for 5–10 mice in each group. *Po0.05, **Po0.01 compared with each control frequency.

non-cholinergic neurons (NANC) nitrergic neurons and endothelial captured as secondary to the hydrolysis reaction.18 This is cells in the erectile tissue results in release of NO that diffuses to consistent with the conclusions that cGMP hydrolysis is fast and surrounding smooth muscle cells where it activates soluble that 50-GMP dissociation is the rate-limiting step of the reaction.18 guanylate cyclase, facilitating the conversion of GTP to cGMP.13 In conclusion, murine corpus cavernosum expresses PDE9 We next investigated the relaxing effect of the PDE9 inhibitor BAY mRNA, and prolonged blockade of PDE9 activity with BAY 73- 73-6691 in mice corpus cavernosum in vitro. Addition of BAY 6691 amplifies the NO-cGMP-mediated cavernosal responses. 73-6691 to the organ bath neither produced corpus cavernosum Therefore, long-term treatment with BAY 73-6691 could be relaxations itself nor significantly potentiated the relaxations beneficial for erectile dysfunction. induced by NaNO2, sildenafil and EFS. However, 21 days treatment with BAY 73-6691 markedly increased the magnitude and CONFLICT OF INTEREST duration of the nitrergic relaxations. BAY 73-6691 also caused The authors declare no conflict of interest. marked leftward shifts in the concentration–response curves to ACh, NaNO2 and sildenafil in the mice corpus cavernosum, suggesting that prolonged PDE9 inhibition acts to amplify the ACKNOWLEDGEMENTS cGMP-mediated cell responses. The reasons why prolonged Fa´bio Henrique da Silva was supported by Coordenaçaõ de Aperfeiçoamento de treatment with BAY 73-6691 (but not in vitro addition) improves Pessoal de Nı´vel Superior (CAPES). Ma´rio Angelo Claudino thanks Fundaçaõde corpus cavernosum relaxations remains to be elucidated. To our Amparo a` Pesquisa do Estado de Saõ Paulo (FAPESP). We are also grateful to Nicola knowledge, this is the first study demonstrating that PDE9 chronic Conran for providing us with BAY 73-6691. inhibition exerts significant effects on relaxing mice CC and requires further study to understand the pharmacodynamics and pharmacokinetics aspects of this drug orally administered. REFERENCES The PDE5 inhibitors generally increase and prolong the 1 Conti M, Jin SLC. The molecular biology of cyclic nucleotide phosphodiesterases. neurogenic NO-mediated cavernosal and ACh-induced relaxations Prog Nucleic Acid Res Mol Biol 2000; 63: 1–38. in animal and human corpus cavernosum, either as oral pretreat- 2 Bender AT, Beavo JA. Cyclic nucleotide phosphodiesterases: molecular regulation 19–25 to clinical use. Pharmacol Rev 2006; 58: 488–520. ment or by in vitro addition. BAY 73-6691 selectively inhibits 3 Kuthe A, Wiedenroth A, Magert HJ, Uckert S, Forssmann WG, Stief CG et al. human and murine PDE9 activity in vitro (IC50 ¼ 55 and 100 nM, Expression of different phosphodiesterase genes in human cavernous smooth respectively) and shows only discrete activity against PDE5 muscle. J Urol 2001; 165: 280–283. 7 (IC5044000 nM) in an ovary cell line. Therefore, it is unlikely that 4 Wang P, Wu P, Egan RW, Billah MM. Identification and characterization of a new enhancement of corpus cavernosum relaxations by BAY 73-6691 human type 9 cGMP-specific phosphodiesterase splice variant (PDE9A5). Differ- reflects a unespecific inhibitory effect at the PDE5 isoform. In ential tissue distribution and subcellular localization of PDE9A variants. Gene 2003; hamster ovary cell line, BAY 73-6691 alone failed to significantly 18: 15–27. increase the basal cGMP levels; however, BAY 73-6691, in 5 Almeida CB, Traina F, Lanaro C, Canalli AA, Saad ST, Costa FF et al. High expression combination with the soluble guanylate cyclase activator (BAY of the cGMP-specific phosphodiesterase, PDE9A, in sickle cell disease (SCD) and the effects of its inhibition in erythroid cells and SCD neutrophils. Br J Haematol 58-2667), concentration dependently enhanced the intracellular 7 2008; 142: 836–844. cGMP accumulation in this cell line. Of interest, under biochemical 6 Nagasaki S, Nakano Y, Masuda M, Ono K, Miki Y, Shibahara Y et al. Phospho- conditions in which PDE9 is fully active, cGMP was cleaved in a few diesterase type 9 (PDE9) in the human lower urinary tract: an immunohisto- seconds, and an enzyme/product (PDE9/50-GMP) complex was chemical study. BJU Int 2012; 109: 934–940.

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