cGMP-dependent mechanism

Accepted Manuscript Title: ␤3 adrenergic receptor activation relaxes human corpus cavernosum and penile artery through a hydrogen sulfide/cGMP-dependent mechanism Authors: Emma Mitidieri, Teresa Tramontano, Danila Gurgone, Ciro Imbimbo, Vincenzo Mirone, Ferdinando Fusco, Giuseppe Cirino, Roberta d’Emmanuele di Villa Bianca, Raffaella Sorrentino PII: DOI: Reference:

S1043-6618(17)30751-X http://dx.doi.org/doi:10.1016/j.phrs.2017.07.025 YPHRS 3656

To appear in:

Pharmacological Research

Received date: Revised date: Accepted date:

22-6-2017 25-7-2017 27-7-2017

Please cite this article as: Mitidieri Emma, Tramontano Teresa, Gurgone Danila, Imbimbo Ciro, Mirone Vincenzo, Fusco Ferdinando, Cirino Giuseppe, di Villa Bianca Roberta d’Emmanuele, Sorrentino Raffaella.␤3 adrenergic receptor activation relaxes human corpus cavernosum and penile artery through a hydrogen sulfide/cGMP-dependent mechanism.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2017.07.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

β3 adrenergic receptor activation relaxes human corpus cavernosum and penile artery through a hydrogen sulfide/cGMP-dependent mechanism

Emma Mitidieria, Teresa Tramontanoa, Danila Gurgonea, Ciro Imbimbob,c, Vincenzo Mironeb,c, Ferdinando Fuscob,c, *Giuseppe Cirinoa,b, #Roberta d’Emmanuele di Villa Biancaa,b, #Raffaella Sorrentinoa,b

a

Department of Pharmacy, University of Naples, Federico II, Via D. Montesano, 49, Naples, Italy,

80131; bInterdepartmental Centre for Sexual Medicine, University of Naples, Federico II, Via Sergio Pansini 5, Naples, Italy, 80131; cDepartment of Neurosciences, Human Reproduction and Odontostomatology, University of Naples, Federico II, Naples, Italy, 80131.

#

The authors have equally contributed

*Corresponding author: Giuseppe Cirino Department of Pharmacy, University of Naples, Federico II, Via Domenico Montesano 49, 80131 Napoli, Italy Tel.: +39 081678442 e-mail: [email protected]

Abstract Erectile function is a widely accepted indicator of systemic endothelial activity since from a clinical standpoint erectile dysfunction (ED) often precedes cardiovascular events. Recently it has been described a potential role for β3 adrenoceptor in cardiovascular diseases emphasizing a possible development of new drugs. β3 adrenoceptor stimulation relaxes human corpus cavernosum (HCC) strips in cyclic guanosine monophosphate (cGMP)-dependent and endothelium/nitric oxide (NO)independent manner. Hydrogen sulfide (H2S), along with NO, is another gaseous molecule involved in cardiovascular system and as a consequence also in penile erection. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), the enzymes mainly responsible for H2S biosynthesis, are constitutively expressed in HCC. CSE rather than CBS is more abundant in human penile tissue. Herein we investigated the involvement of H2S pathway in β3 adrenoceptor-induced relaxation in HCC and penile artery. Penile artery expresses both CSE and β3 adrenoceptor. BRL37344, a β3 selective agonist, relaxed HCC strips and penile artery rings and this effect was significantly reduced by CSE inhibition. Incubation of HCC and penile artery homogenate with BRL37344 significantly increased H2S production. This effect was significantly reduced by the inhibition of either CSE or β3 adrenoceptor. Finally, the BRL37344-induced increase in cGMP was reduced by CSE inhibition in both tissues. Thus, BRL37344-induced relaxation in HCC and penile artery occurs in a H2S/cGMP-dependent manner. In conclusion, β3/H2S/cGMP pathway can act as an alternative to NO. Since about 15% of patients do not respond to phosphodiesterase-5 inhibitors, β3 agonists could represent a therapeutic alternative or a useful adjuvant therapy to treat these patients.

Key words: β3 adrenoceptor, cGMP, human corpus cavernosum, human penile artery, hydrogen sulfide, relaxation

Abbreviations CBS, cystathionine β-synthase cGMP, cyclic guanosine monophosphate CSE, cystathionine-γ-lyase ED, erectile dysfunction eNOS, endothelial nitric oxide synthase H2S, hydrogen sulfide HCC, human corpus cavernosum NO, nitric oxide PAG, DL-propargylglycine PDE5, phosphodiesterase -5 PE, phenylephrine sGC, soluble guanylate cyclase

Chemical compounds studied in this article: BRL37344, Hydrogen sulfide

1. Introduction Penile erection is strictly dependent upon vascular tone. Indeed, erection is reached through a coordinated relaxation of arteries resulting in sinusoid expansion and increased intracavernous pressure [1,2]. A central role in the transduction mechanism in erection is played by the L-argininenitric oxide (NO) pathway. NO, by stimulating the soluble guanylate cyclase (sGC) leads to an increase in cyclic guanosine monophosphate (cGMP) production [3]. Therefore, an increase in cGMP in the human corpus cavernosum (HCC) leads to an improvement of penile erection implying a central role for cGMP in the human erectile mechanism(s), as confirmed by the clinical use of phosphodiesterase -5 (PDE5) inhibitors. Indeed, this class of drugs is considered a mainstay in the erectile dysfunction (ED) therapy. Moreover, β3 adrenoceptors are, among the different receptors and mediators, identified within the corpus cavernosum [4,5]. The β3 adrenoeceptor contributes to penile erection since in vitro its activation causes relaxation of HCC isolated strips in a cGMP-dependent and NO-independent manner [5]. Recently, mirabegron, a commercially available selective β3 agonist, approved for the treatment of overactive bladder, has been shown to cause a concentration-dependent relaxation of human and rat corpus cavernosum [4]. However, how β3 adrenoceptor stimulation leads to an NO-independent-cGMP increase in penile tissues is still unclear. Hydrogen sulfide (H2S) is a gaseous transmitter constitutively produced in mammalian tissue from the substrate L-cysteine [6-9]. H2S synthesis mainly involves two pyridoxal-dependent enzymes i.e. cystathionine β-synthase (CBS) and cystathionine-γ-lyase (CSE) [10-13]. H2S is considered one of endogenous mediator involved in the human penile erectile mechanism. Indeed, either H2S donors or L-cysteine, an endogenous substrate, induces HCC relaxation [14]. Both CBS and CSE are expressed in HCC. In particular, CSE is selectively localized on peripheral nerves while on the muscular trabeculae and smooth muscle also CBS is expressed but a lesser extent than CSE [14]. For what concerns the molecular mechanisms it has been demonstrated that H2S can increase cGMP accumulation at vascular level either directly by increasing intracellular

NO bioavailability [15], indirectly by inhibiting the enzymatic activity of PDE [16] or reducing sGC heme Fe thereby facilitating the NO-mediated cellular signaling [17,18]. This link between cGMP and H2S is further stressed by the finding that either sildenafil or a stable analogue of cGMP, can significantly increase H2S production in human bladder [19,20]. The aim of the present study is to evaluate the involvement of H2S pathway in cGMP elevation and relaxation following β3 adrenoceptor stimulation in human penile tissue.

2. Materials and Methods 2.1. Human tissue In male to female transsexuals undergoing surgical procedure for sex reassignment, the penis and testicles were amputated and a neo-vagina was created to simulate female external genitalia. All the surgical procedures were performed at the Department of Neurosciences, Human Reproduction and Odontostomatology, University of Naples, Federico II, Naples, Italy, 80131. The corpora cavernosa were carefully excised from the penis immediately after amputation and placed in an ice-cold oxygenated Krebs’ solution [21]. The research has been carried out in accordance with the Declaration of Helsinki (2013) of the World Medical Association. The Ethical Committee of the Institution (School of Medicine and Surgery, University of Naples Federico II, via Pansini, 5; 80131, Naples, Italy) in which the study was performed, has approved it. The subjects have given written informed consent to the work.

2.2. HCC Strips Longitudinal strips (2 cm) of HCC were dissected and isolated from the trabecular structure of the penis [22]. Krebs solution had the following composition (mM): 115.3 NaCl; 4.9 KCl; 1.46 CaCl 2; 1.2 MgSO4; 1.2 KH2PO4; 25.0 NaHCO3; 11.1 glucose (Carlo Erba, Milan, Italy). HCC strips were mounted in organ bath containing oxygenated (95% O2 and 5% CO2) Krebs solution at 37°C. HCC strips were connected to isometric force-displacement transducers (model 7002, UgoBasile, Comerio, Italy) and changes in tension were recorded continuously by using a data acquisition system (Data Capsule 17400, Ugo Basile, Varese, Italy). Tissues were preloaded with 2 g of tension and allowed to equilibrate for 90 minutes in Krebs solution. After equilibration, tissues were standardized by performing repeated phenylephrine (PE; 1µM; Sigma, Milan, Italy) contractions until three equal responses were obtained. A concentration-response curve to BRL37344, a β3 selective adrenoceptor agonist (10 µM–300 µM, Tocris, UK) was obtained on strips pre-contracted

with PE (1 µM). Thereafter, the strips were incubated for 30 minutes with DL-propargylglycine (PAG; 10 mM; Sigma, Milan, Italy), as CSE inhibitor, before BRL37344 challenge.

2.3. Human penile artery Penile artery, dissected and isolated from HCC, was cut in rings (2-3 mm) and mounted in organ bath containing oxygenated (95% O2 and 5% CO2) Krebs solution at 37°C. Penile artery rings were connected to isometric force-displacement transducers (model 7010C, Ugo Basile, Comerio, Italy) and changes in tension were continuously recorded by using a data acquisition system (PowerLab/800, 2biological Instruments, Varese, Italy). Tissues were preloaded with 1 g of tension and allowed to equilibrate for 60 minutes in Krebs solution. After equilibration, tissues were standardized by performing repeated PE contractions (3 µM) until three equal responses were obtained. A concentration-response curve to BRL37344 (10 µM–300 µM) was obtained on rings pre-contracted with PE (3 µM). After that, the same rings were incubated with PAG (10 mM, 30 minutes) and the concentration-response curve with BRL37344 was repeated.

2.4. Western blot Western blot was performed as previously described [23]. Briefly, frozen human penile artery was homogenized in modified RIPA buffer (Tris-HCl 50 mM pH 8.0, NaCl 150 mM, sodium deoxycholate 0.5%, sodium dodecyl sulphate 0.1%, EDTA 1 mM, Igepal 1%) (Roche Applied Science, Italy) and protease inhibitor cocktail (Sigma, Milan, Italy). Protein concentration was determined by Bradford assay using albumin (BSA, Sigma, Milan, Italy) as standard (Sigma, Milan, Italy). Denatured proteins (50 µg) were separated on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to a polyvinylidene fluoride membrane. The membranes were blocked by incubation in phosphate buffer solution (PBS) containing 0.1% v/v Tween 20 and 5% non-fat dried milk for 1 h at room temperature and then incubated with mouse monoclonal antibody for CSE (1:1000; Abnova, Milan, Italy) or rabbit polyclonal antibody for β3 (1:1000; Novus Biological,

Cambridge, UK) overnight at 4°C. Membranes were extensively washed in PBS containing 0.1% v/v Tween-20 prior to incubation with horseradish peroxidase-conjugated secondary antibody for 2h at room temperature. Following incubation, membranes were washed and developed using ImageQuant-400 apparatus (GE Healthcare, USA). The target protein band intensity was normalized over the intensity of the house keeping protein ß-actin (1:5000, Sigma-Aldrich, Milan, Italy).

2.5. H2S determination HCC strips were incubated with vehicle or BRL37344 at different concentrations (0.1, 1, 10, 100 µM) for 30 minutes and thereafter the concentration of 10 µM was chosen for the following experiments. As well samples of penile artery were incubated with vehicle or BRL37344 (10 µM) for 30 minutes. HCC or penile arterial samples were incubated for 20 minutes with PAG (10 mM), a CSE inhibitor, or SR59230A (10 µM; Sigma, Milan, Italy), a β3 selective antagonist and then stimulated with BRL37344 (10 µM). All the samples were frozen and the H2S level was measured as previously reported [23,24]. Briefly, samples were lysed in an appropriated buffer (potassium phosphate buffer 100 mM, pH 7.4, sodium orthovanadate 10 mM, and proteases inhibitors). Protein concentration was determined by using Bradford assay (Bio-Rad Laboratories). The reaction was performed in sealed eppendorf tubes and initiated by transferring tubes from ice to a water bath at 37 °C for 30 min. Next, trichloroacetic acid solution (10% wt/vol) was added to each sample followed by zinc acetate (1% wt/vol). Subsequently, N,N-dimethyl-p-phenylendiaminesulfate (DPD; 20 mM) in HCl (7.2 M) and FeCl3 (30 mM) in HCl (1.2 M) were added and optical absorbance of the solutions was measured after 20 min at a wavelength of 668 nm. All samples were assayed in duplicate, and H2S concentrations were calculated against a calibration curve of NaHS (3–250 μM).

2.6. cGMP measurement In order to measure cGMP content, HCC or human penile artery samples were stimulated with BRL37344 (10 µM, for 30 minutes) or vehicle as control, respectively. In another setting of experiments, samples were pre-treated with PAG (10 mM, for 20 minutes) and then stimulated with BRL37344. Thereafter the reaction was stopped with liquid nitrogen. Samples were then dropped into 5-10 volumes (ml of buffer/g of tissue) of TCA (5%) and homogenized by using a polytrontype homogenizer. Samples were centrifuged at 1500×g for 10 minutes and cGMP was measured in supernatants as described in the manufactures protocol of cGMP EIA Kit (Cayman, Vinci Biochem, Vinci, Italy) [25,26].

3.Statistical analysis Data were analyzed by using one way ANOVA or two way ANOVA following by Bonferroni as post -test, as needed. p<0.05 was considered significant.

4. Results 4.1. HCC strips The ß3 selective agonist BRL37344 (10 µM–300 µM), as also previously shown, relaxed HCC strips in a concentration-dependent manner (Figure 1). In order to evaluate the involvement of the H2S pathway we used PAG, a CSE inhibitor. Incubation of HCC strips with PAG (10 mM) significantly reduced BRL37344 relaxation (***p<0.001, Figure 1). PAG at the concentration used did not alter PE-induced contraction (data not shown). Homogenates of HCC generated a detectable amount of H2S (Figure 2A). BRL37344 significantly increased H2S production, reaching its maximum at 10 µM (***p<0.01, Figure 2A). The blockade of either CSE with PAG (10 mM) or β3 adrenoceptor with the selective antagonist SR59230A (10 µM) significantly reduced BRL37344-induced increase in H2S production (°°p<0.01, Figure 2B).

4.2. Penile artery Western blot analysis clearly demonstrated that β3 adrenoceptor and CSE were expressed in human penile artery (Figure 3A). BRL37344 (10 µM–300 µM) relaxed human penile artery rings precontracted with PE in a concentration-dependent manner (Figure 3B). The incubation with PAG (10 mM), a CSE inhibitor, significantly reduced BRL37344-induced relaxation (**p<0.01; Figure 3B). To further confirm the involvement of the H2S pathway, a colorimetric assay was also performed on human penile artery samples. A detectable amount of H2S was found in the human penile artery in basal condition. BRL37344 (10 µM) increased H2S production by about 3 fold (**p<0.01, Figure 3C). PAG (10 mM), a CSE inhibitor or SR59230A (10 µM), a selective β3 adrenoceptor antagonist, significantly inhibited BRL37344-induced increase in H2S production (°p<0.05, Figure 3C).

4.3. cGMP measurement in HCC and penile artery Incubation of HCC tissue with BRL37344 (10 µM) increased cGMP production (*p<0.05 vs vehicle; Figure 4A). The inhibition of CSE with PAG significantly reduced the BRL37344-induced

increase in cGMP content (°p<0.05; Figure 4A). Similarly, incubation of penile artery tissue with BRL37344 (10 µM) significantly increased cGMP levels (**p<0.01 vs vehicle; Figure 4B). The pre-treatment with PAG (10 mM) reversed the BRL37344-induced effect (°°p<0.01; Figure 4B).

5. Discussion β3 adrenoceptor has recently come into focus after its clear identification in human cardiovascular tissues [27]. Its particular properties and coupling in heart and vessels, as well as other tissues (e.g. brown fat, bladder) has shed new light on the catecholaminergic regulation in cardiovascular system. The vascular β3-adrenoceptor are stimulated by very high doses of catecholamines implying that are activated during extreme or stressful conditions while in pathological conditions the expression of these receptors is increased [27]. In the vasculature, the expression of β3 adrenoceptors is different between vascular beds, depending upon vessel’s caliber and species [27]. The transduction mechanism underlying activation of β3 adrenoceptors has been widely investigated in experimental animal studies while there are still few human data [28]. β3 adrenoceptors are expressed in HCC and their activation leads to smooth muscle relaxation in a cGMP-dependent and NO-independent manner [5]. Indeed, the relaxing effect of BRL37344 is not affected either by the inhibition of endothelial nitric oxide synthase (eNOS) or NO-sensitive GC [5]. However, this effect is associated to an increase in cGMP level. The new selective β3 agonist, mirabegron, approved for the treatment of overactive bladder, also causes an endothelium and NO- independent relaxation of human and rat corpus cavernosum [4]. Thus, since NO does not account for the increase in cGMP we have evaluated the possible involvement of the H2S pathway in β3 signaling in HCC and in human penile artery. The idea to select H2S as a feasible candidate is supported by the findings that i) H2S, by acting as an endogenous PDE inhibitor, increases cGMP levels and ii) sildenafil, a PDE-5 inhibitor, increases H2S production in the human bladder [14,16,19]. Since CSE is the main source of H2S in HCC [15], we performed a relaxation curve with BRL37344 in presence of PAG, a CSE inhibitor. PAG caused a significant inhibition of β3 induced- relaxation of HCC strips whit a marked shift of the EC50 from 80±1.51 µM to 124±1.58 µM. BRL37344 also increased H2S generation in HCC tissue homogenate. These data clearly indicate the involvement of CSE-derived H2S in the β3 effect. We confirmed that this effect was indeed β3 adrenoceptor-dependent by using a selective β3 antagonist

SR59230A. Of particular interest is the finding that PAG or SR59230A inhibited the H2S release in vitro at the same extent further suggesting a key role for H2S pathway following β3 adrenoceptor activation. It has been demonstrated that H2S acts as PDE inhibitor causing an increase in cGMP level [17] and that β3 adrenoceptor activation in HCC leads to an increase in cGMP in a NO-independent manner [3]. In order to further define a link between the CSE/H2S pathway and the β3 activation we have evaluated if changes in cGMP levels induced by BRL37344 could be modulated through CSE inhibition. Incubation with CSE inhibitor reversed the effect given by BRL37344 bringing back the cGMP levels to the basal value. Since the HCC strips is not a pure vascular preparation and since it is known that the β3 effect is strictly linked to vessel caliber we have investigated if the same mechanism was present in the penile artery. Penile artery tone plays a key role in the erection mechanism that leads to the increase in blood pressure within the corpora cavernosa. Therefore it represents also a relevant tissue to study the physiopathology of the penis. We demonstrate that human penile artery expresses both the β3 adrenoceptors and CSE. The functional study, performed on isolated penile artery rings, demonstrated that BRL37344 caused a concentration-dependent relaxation. The relaxant effect was inhibited by CSE blockade at the same extent and with a similar profile as compared to HCC strips. It is feasible that following β3 adrenoceptor stimulation the generated H2S derives from the smooth muscle cells since CSE is mainly located in muscle component of either corpus cavernosum or penile artery in human as previously demonstrated [14]. Similarly to HCC tissue, incubation of penile artery tissue homogenate with the β3 agonist caused a significant increase in H2S level that was inhibited either by PAG or SR59230A. Interestingly both PAG and SR59230A reversed the effect of β3 agonist bringing back the H2S value to the basal levels. The incubation of penile artery tissue homogenate with BRL37344 leads to an increase in cGMP level of about three fold as compared to the basal value. CSE blockade by PAG also in this case reversed BRL37344 effect. Therefore, β3 activation either in HCC or penile artery leads to an increase of CSE-derived H2S

which in turn enhances cGMP levels. This effect is explained by the finding that H2S inhibits PDE activity [17]. Therefore, β3 receptors present on HCC and on the vasculature can contribute to penile erection through a cGMP/CSE/H2S dependent mechanism. The NO/cGMP signaling is the main metabolic pathway leading to penile erection in men and also the main pharmacological target for the treatment of ED. Indeed, PDE-5 inhibitors represent the first line of treatment for ED. Nonetheless, clinical data highlight that 15% of patients do not respond to this therapy. This lack of effect has been generally ascribed to the presence of a high degree of endothelial dysfunction. On this basis, since β3 agonist do not need of endothelium to cause relaxation, it could represent an alternative therapy in patients not fully responding to PDE-5 inhibitors alone, especially those with ED associated to neuronal and/or endothelial damage.

6. Conclusion The mechanism(s) of downstream β3 adrenoceptor activation in HCC and in penile artery involves CSE-derived H2S and cGMP elevation. In other words, β3 relaxation does not need the endothelium but is still able to amplify the cGMP signaling through H2S pathway. Therefore, this study defines a possible novel pharmacological approach in ED treatment and could also improve the patient general vascular condition.

Funding This work was supported by Interdepartmental Centre for Sexual Medicine (CIRMS) structural funding and by Regione Campania under POR Campania FESR 2007-2013 - O.O. 2.1 (FarmaBioNet).

Figure 1. BRL37344 relaxed HCC strips through CSE-H2S derived. BRL37344 (10 µM-300 µM) relaxed HCC strips in a concentration-dependent manner. CSE inhibitor (PAG 10 mM) significantly reduced BRL37344-induced relaxation (***p<0.001). Data were calculated as percent of relaxation to PE tone and expressed as mean±SEM from five separate specimens from five different tissues.

Figure 2. BRL37344 modulates H2S production in HCC homogenates. BRL37344 (0.1 µM-100 µM) caused an increase in H2S production compared to vehicle (Panel A; ***p<0.001). PAG (10 mM), a CSE inhibitor or SR59230A (10 µM), a β3 antagonist, significantly inhibited BRL37344-induced H2S production in HCC (Panel B;°°p<0.01, versus BRL37344 alone). Data were expressed as mean±SEM from five separate specimens from five different tissues.

Figure 3. BRL37344 relaxed human penile artery through CSE-H2S derived. Western blot analysis for β3 receptor and CSE in human penile artery. β-actin was used as loading control (Panel A). BRL37344 (10 µM-300 µM) relaxed penile artery rings in a concentrationdependent manner. BRL37344-induced relaxation was significantly reduced by PAG (10 mM; **p<0.01). Results were calculated as percent of relaxation to PE tone (Panel B). BRL 37344 (10 µM) caused a significant increase in H2S production compared to vehicle (**p<0.01). PAG (10 mM), a CSE inhibitor, or SR59230A (10 µM), a β3 antagonist, significantly inhibited BRL37344induced H2S production (°p<0.05) (Panel C). Data were expressed as mean±SEM from five separate specimens from five different tissues.

Figure 4. BRL37344 increased cGMP levels through CSE activation in HCC and human penile artery. BRL37344 (10 µM) significantly increased cGMP levels compared with vehicle in HCC (*p<0.05). PAG (10 mM) reversed BRL37344-induced effect (°p<0.05) (Panel A). BRL37344 (10 µM)

significantly increased cGMP levels compared with vehicle in human penile artery (**p<0.01). PAG (10 mM) reversed BRL37344-induced effect (°°p<0.01) (Panel B). Data were expressed as mean±SEM for five separate specimens from five different tissues.

References [1] A.L. Burnett, Role of nitric oxide in the physiology of erection, Biol Reprod. 52 (1995) 485-9. [2] M.A. Khan, R.J. Morgan,D.P. Mikhailidis, The molecular basis of penile erection, Curr Med Res Opin. 16 Suppl 1 (2000) s21-30. [3] G. Yetik-Anacak, R. Sorrentino, A.E. Linder,N. Murat, Gas what: NO is not the only answer to sexual function, Br J Pharmacol. 172 (2015)1434-54. [4] S. Gur, T. Peak, F.A. Yafi, P.J. Kadowitz, S.C. Sikka,W.J. Hellstrom, Mirabegron causes relaxation of human and rat corpus cavernosum: could it be a potential therapy for erectile dysfunction?, BJU Int. 118 (2016) 464-74. [5] G. Cirino, R. Sorrentino, R. Di Villa Bianca, A. Popolo, A. Palmieri, C. Imbimbo, F. Fusco, N. Longo, G. Tajana, L.J. Ignarro,V. Mirone, Involvement of beta 3-adrenergic receptor activation via cyclic GMP- but not NO-dependent mechanisms in human corpus cavernosum function, Proc Natl Acad Sci U S A. 100 (2003) 5531-6. [6] H. Kimura, Hydrogen sulfide: its production, release and functions, Amino Acids. 41 (2011) 113-21. [7] R. Wang, The gasotransmitter role of hydrogen sulfide, Antioxid Redox Signal. 5 (2003) 493501. [8] M.M. Gadalla,S.H. Snyder, Hydrogen sulfide as a gasotransmitter, J Neurochem. 113 (2010) 1426. [9] P. Kamoun, Endogenous production of hydrogen sulfide in mammals, Amino Acids. 26 (2004) 243-54. [10] R. Wang, Physiological implications of hydrogen sulfide: a whiff exploration that blossomed, Physiol Rev. 92 (2012) 791-896. [11] C. Szabo, Hydrogen sulphide and its therapeutic potential, Nat Rev Drug Discov. 6 (2007) 917-35.

[12] H. Kimura, Production and physiological effects of hydrogen sulfide, Antioxid Redox Signal. 20 (2014) 783-93. [13] L. Li, P. Rose,P.K. Moore, Hydrogen sulfide and cell signaling, Annu Rev Pharmacol Toxicol. 51 (2011) 169-87. [14] R. d'Emmanuele di Villa Bianca, R. Sorrentino, P. Maffia, V. Mirone, C. Imbimbo, F. Fusco, R. De Palma, L.J. Ignarro,G. Cirino, Hydrogen sulfide as a mediator of human corpus cavernosum smooth-muscle relaxation, Proc Natl Acad Sci U S A. 106 (2009) 4513-8. [15] C. Coletta, A. Papapetropoulos, K. Erdelyi, G. Olah, K. Modis, P. Panopoulos, A. Asimakopoulou, D. Gero, I. Sharina, E. Martin,C. Szabo, Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation, Proc Natl Acad Sci U S A. 109 (2012) 9161-6. [16] M. Bucci, A. Papapetropoulos, V. Vellecco, Z. Zhou, A. Pyriochou, C. Roussos, F. Roviezzo, V. Brancaleone,G. Cirino, Hydrogen sulfide is an endogenous inhibitor of phosphodiesterase activity, Arterioscler Thromb Vasc Biol. 30 (2010) 1998-2004. [17] C. Szabo, Hydrogen sulfide, an enhancer of vascular nitric oxide signaling: mechanisms and implications, Am J Physiol Cell Physiol. 312 (2017) C3-C15. [18] Z. Zhou, E. Martin, I. Sharina, I. Esposito, C. Szabo, M. Bucci, G. Cirino,A. Papapetropoulos, Regulation of soluble guanylyl cyclase redox state by hydrogen sulfide, Pharmacol Res. 111 (2016) 556-62. [19] F. Fusco, R. d'Emmanuele di Villa Bianca, E. Mitidieri, G. Cirino, R. Sorrentino,V. Mirone, Sildenafil effect on the human bladder involves the L-cysteine/hydrogen sulfide pathway: a novel mechanism of action of phosphodiesterase type 5 inhibitors, Eur Urol. 62 (2012) 1174-80. [20] R. d'Emmanuele di Villa Bianca, E. Mitidieri, D. Esposito, E. Donnarumma, A. Russo, F. Fusco, A. Ianaro, V. Mirone, G. Cirino, G. Russo,R. Sorrentino, Correction: Human Cystathioninebeta-Synthase Phosphorylation on Serine227 Modulates Hydrogen Sulfide Production in Human Urothelium, PLoS One. 10 (2015) e0141027.

[21] R. d'Emmanuele di Villa Bianca, G. Cirino, E. Mitidieri, C. Coletta, G. Grassia, F. Roviezzo, P. Grieco, E. Novellino, C. Imbimbo, V. Mirone,R. Sorrentino, Urotensin II: a novel target in human corpus cavernosum, J Sex Med. 7 (2010) 1778-86. [22] R. d'Emmanuele di Villa Bianca, E. Mitidieri, F. Fusco, E. D'aiuto, P. Grieco, E. Novellino, C. Imbimbo, V. Mirone, G. Cirino,R. Sorrentino, Endogenous urotensin II selectively modulates erectile function through eNOS, PLoS One. 7 (2012) e31019. [23] R. d'Emmanuele di Villa Bianca, E. Mitidieri, E. Donnarumma, T. Tramontano, V. Brancaleone, G. Cirino, M. Bucci,R. Sorrentino, Hydrogen sulfide is involved in dexamethasoneinduced hypertension in rat, Nitric Oxide. 46 (2015) 80-6. [24] E. Mitidieri, T. Tramontano, E. Donnarumma, V. Brancaleone, G. Cirino, R. d'Emmanuele di Villa Bianca,R. Sorrentino, l-Cys/CSE/H2S pathway modulates mouse uterus motility and sildenafil effect, Pharmacol Res. 111 (2016) 283-9. [25] V. Mirone, R. d'Emmanuele di Villa Bianca, E. Mitidieri, C. Imbimbo, F. Fusco, P. Verze, D.F. Vitale, R. Sorrentino,G. Cirino, Platelet cyclic guanosine monophosphate as a biomarker of phosphodiesterase type 5 inhibitor efficacy in the treatment of erectile dysfunction: a randomized placebo-controlled study, Eur Urol. 56 (2009) 1067-73. [26] R. d'Emmanuele di Villa Bianca, E. Mitidieri, V. Mirone, F. Fusco, C. Imbimbo, G. Cirino,R. Sorrentino, An ex vivo standardized assay to measure human platelet cGMP, J Pharmacol Toxicol Methods. 64 (2011) 164-7. [27] B. Rozec,C. Gauthier, beta3-adrenoceptors in the cardiovascular system: putative roles in human pathologies, Pharmacol Ther. 111 (2006) 652-73. [28] J.L. Balligand, Cardiac salvage by tweaking with beta-3-adrenergic receptors, Cardiovasc Res. 111 (2016) 128-33.