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ORIGINAL RESEARCH—BASIC SCIENCE. Effect of Chronic Tadalafil Administration on Penile Hypoxia. Induced by Cavernous Neurotomy in the Rat.
jsm_208.fm Page 419 Wednesday, April 12, 2006 12:11 PM Blackwell Publishing IncMalden, USAJSMJournal of Sexual Medicine1743-6095© 2006 International Society for Sexual Medicine200633419431Original ArticleNeurotomy-Induced Hypoxia and Chronic TadalafilVignozzi et al.

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ORIGINAL RESEARCH—BASIC SCIENCE Effect of Chronic Tadalafil Administration on Penile Hypoxia Induced by Cavernous Neurotomy in the Rat Linda Vignozzi, MD,* Sandra Filippi, PhD,† Annamaria Morelli, PhD,* Stefano Ambrosini, PhD,‡ Michaela Luconi, PhD,* Gabriella Barbara Vannelli, MD,‡ Silvia Donati, PhD,* Clara Crescioli, PhD,* Xin-Hua Zhang, MD,* Vincenzo Mirone, MD,§ Gianni Forti, MD,* and Mario Maggi, MD* *Andrology Unit, Department of Clinical Physiopathology, Center of Research, Transfer and High Education, † Interdepartmental Laboratory of Functional and Cellular Pharmacology of Reproduction, Departments of Pharmacology and Clinical Physiopathology, and ‡Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy; §Department of Urology, University of Medicine “Federico II,” Naples, Italy DOI: 10.1111/j.1743-6109.2006.00208.x

ABSTRACT

Background. Numerous men develop postprostatectomy erectile dysfunction (PPED), due to surgery-related nervous damage. PPED is often refractory to phosphodiesterase type 5 (PDE5) inhibitors therapy. Aim. To verify whether chronic tadalafil (CT) preserves bilateral cavernous neurotomy (BCN)-induced penile damage and hypo-oxygenation. Methods. In a rat model of BCN we evaluated in vitro and ex vivo effect of CT treatment (2 mg/kg, daily for 3 months). Results. Bilateral cavernous neurotomy induced massive hypoxia and decreased muscle/fiber ratio, completely restored by CT. Hypersensitivity of hypoxic tissues to the relaxant effect of the endothelin type B receptor (ETB) agonist IRL-1620 was observed, along with ETB mRNA and protein overexpression. CT restored sensitivity to IRL-1620, and normalized ETB expression. Hypoxic penis showed increased sensitivity to the relaxant effect of the nitric oxide donor sodium nitroprusside (SNP), while acute tadalafil (100 nM) did not amplify the SNP effect. Accordingly, PDE5 mRNA and protein were reduced in BCN penile tissues. By restoring PDE5, CT decreased SNP-induced relaxation and rescued sensitivity to acute tadalafil. However, in hypoxic penis, CT normalizes neither acetylcholine hyporesponsiveness nor neuronal nitric oxide synthase-endothelial nitric oxide synthase expression. Conclusion. Chronic tadalafil restores some of the investigated BCN-induced alterations, including PDE5 and tadalafil efficacy. Vignozzi L, Filippi S, Morelli A, Ambrosini S, Luconi M, Vannelli GB, Donati S, Crescioli C, Zhang X-H, Mirone V, Forti G, and Maggi M. Effect of chronic tadalafil administration on penile hypoxia induced by cavernous neurotomy in the rat. J Sex Med 2006;3:419–431. Key Words. Animal Models; Pelvic Cancer Treatments and Sexual Dysfunction; Pharmacological Studies in Sexual Function; Molecular Biological Studies of Sexual Function

Introduction

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rostate cancer is one of the most common male malignancies, which, in its localized form, is usually treated by radical retropubic prostatectomy (RRP). After RRP, even following the introduction of nerve-sparing techniques [1], postprostatectomy erectile dysfunction (PPED) remains a persistent, severe problem for most © 2006 International Society for Sexual Medicine

patients [2–6], often crucial to men’s self-esteem and quality of life. In addition, in PPED, responsiveness to oral phosphodiesterase type 5 (PDE5) inhibitors (PDE5i) was variable, but overall lower than in patients with other forms of erectile dysfunction [7–10]. Nonetheless, after the introduction of Walsh’s technique of nerve preservation, the rate of postprostatectomy sexual recovery greatly improved. This strongly indicates that surJ Sex Med 2006;3:419–431

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420 gery-related nerve injury, with the consequent degeneration of cavernous nerves, plays a major role in PPED. In fact, cavernous nerves supply the important autonomic signals needed for erection induction. Bilateral cavernous neurotomy (BCN) in the rat is a well-established model to study effects of PPED on penile tissue [11,12]. In this model it has been demonstrated that cavernous nerve injury is coupled to precocious apoptosis of penile smooth muscle cells and persistent penile atrophy [13,14]. Accordingly, even after long-term (12–15 weeks) cavernous nerve injury, an increase in collagen/smooth muscle ratio and transforming growth factor (TGF)-β1 expression was recorded, along with an increase in hypoxia inducible factor (HIF)-1α, a transcriptional factor strictly related to hypoxia [13,15,16]. Hence, it is generally assumed, but never directly demonstrated, that cavernous neurotomy-induced penile injury is coupled to prolonged hypoxia [17]. Hypoxia is a rather physiological event in the penis, which, in the flaccid state, resides at a very low oxygen tension (PO2 = 25–40 mm Hg) [18]. However, in normal men, penile hypoxia is daily interrupted by discrete (total 1.5–3 hours), sleeprelated, erectile episodes [19], which substantially reoxygenate the penis (PO2 = 90–100 mm Hg). The lack of these daily erectile episodes, because of cavernous nerve injury, might underlie the neurotomy-induced penile atrophy and fibrosis [17]. Accordingly, we reported that in human penile smooth muscle cell (hPSMC) culture prolonged (more than 24 hours) hypoxia resulted in an increased endothelin-1 (ET-1) production and activity [20]. ET-1 is a profibrotic peptide [21], whose synthesis in the penis is TGF-β1dependent [20]. In addition, as originally demonstrated by Saenz de Tejada et al. [22] and confirmed in subsequent studies [20,23–25], ET-1 is the most potent stimulator of penile smooth muscle cell contractility through the endothelin type A (ETA). Interestingly, we previously found by in vitro [20] and ex vivo [23] experiments that penile prolonged (more than 24 hours) hypoxia was associated with the upregulation of the vasorelaxant ETB receptor subtype, probably in order to counteract the contractile ET-1 effects. In the past few years, there has been a growing interest in penile rehabilitation after RRP to prevent—or decrease—the incidence of PPED and to increase the low responsiveness rate to PDE5i [5]. Montorsi and colleagues first employed successfully intracorporeal injections of alprostadil two to three times per week with the aim to stimulate J Sex Med 2006;3:419–431

Vignozzi et al. penile tumescence and oxygenation [26]. Later on, early, daily based, administration of PDE5i was suggested, with the purpose to increase nitric oxide (NO) signaling and therefore vasodilatation and oxygenation. Preliminary results with 100 mg sildenafil, a PDE5i, were somehow encouraging, being statistically significantly better than those obtained with placebo [27]. However, the overall rate of return to normal erectile function in that study was only 27% [27]. Another recent report indicates that early use of 100 mg sildenafil every other day was able to preserve the loss of penile smooth muscle content [28], a critical step in the pathogenesis of permanent erectile dysfunction [29]. The aim of the present study was to evaluate morphological and biochemical alterations in rat penile tissue after long-term (3 months) BCN, a lag time compatible with the spontaneous sexual recovery described in humans after RRP (12– 24 months). The effect of a chronic treatment with oral tadalafil (2 mg/kg in drinking water) was also studied. We previously demonstrated in the rat the lack of tachyphylaxis and the effectiveness of chronic tadalafil (CT) dosing, when compared with an acute tadalafil administration (6 mg/kg), in terms of penile erection induced by electrostimulation of the cavernous nerve [30]. Results from this study demonstrate that CT administration allows tissue oxygenation and restores several, but not all, of the investigated neurotomy-induced penile damages. Materials and Methods

Chemicals Phenylephrine HCl (Phe), acetylcholine (Ach), sodium nitroprusside (SNP), N w-nitro-L-arginine methyl ester hydrochloride (L-NAME), reagents for immunocytochemistry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), phosphate-buffered saline (PBS), bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). ET-1 and the ETB selective agonist Suc-(Glu [9], Ala [11,15])-endothelin1 (IRL-1620) were purchased from Novabiochem (Switzerland). Hypoxyprobe™-1 Kit for the detection of tissue hypoxia was purchased from Chemicon International (Temecula, CA, USA). Anti-human ETB receptors, purified rabbit immunoglobulin G (IgG) affinity, were purchased from Assay Design, Inc. (Ann Arbor, MI, USA). Antiendothelial nitric oxide synthase (eNOS) antibody was from Santa Cruz Biotechnology (Santa Cruz,

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Neurotomy-Induced Hypoxia and Chronic Tadalafil CA, USA), anti-neuronal nitric oxide synthase (nNOS) antibody was from BD Bioscence (San Jose, CA, USA), anti-PDE5 antibody was a kind gift of Professor M. Giorgi (Department of Experimental Medicine, University of L’Aquila, Italy). The BM-enhanced chemiluminescence system was purchased from Roche Diagnostic (Milan, Italy). The protein measurement kit was from Bio-Rad Laboratories, Inc. (Hercules, CA).

Animals and Tissues Male adult Sprague–Dawley rats (Harlan, San Pietro al Natisone, Italy) weighing 275–330 g were used. Male rats were separated into three groups: sham-operated, BCN, and BCN plus CT therapy. Tadalafil has been administered orally, dissolved in drinking water (2 mg/kg, daily for 3 months). The animal house technicians controlled every day that animals totally drunk the respective daily dosage of water + tadalafil. In a previous publication [30] we have demonstrated that in the rat this way of chronic administration of tadalafil sorted similar erectile effects (after electrostimulation of the cavernous nerve) to an acute administration of tadalafil (6 mg/kg by oral gavage, 2 hours proceding the experiment). Animals were anesthetized with sodium pentobarbital (50 mg/kg) intraperitoneally injected. Operations were performed under aseptic conditions. A lower abdominal midline incision was made from symphysis pubis to mid abdomen. The testes were retracted, their gubernacula were divided, and they were packed into the upper abdomen. The dorsal lobes of the prostate were exposed. The cavernous nerves were identified, dissected, and transected on both sides. For shamoperated controls, the cavernous nerves were identified but not cut. The wound was closed in a two-layer fashion with interrupted suture. Corpora cavernosa (CC) samples from each experimental group were collected and processed for the following in vitro studies. In Vitro Contractility Studies As previously reported [23], rat CC strips were attached to an isometric muscle bath system and equilibrated for at least 60 minutes at 37°C in Krebs-bicarbonate solution containing (in mM) NaCl 118.1, KCl 4.7, KH2PO4 1.0, MgSO4 1.0, NaHCO3 25.0, CaCl2 2.5, and glucose 11.1 in bath chamber, gassed with 95% O2 and 5% CO2. Tissue-resting force was set at 500 mg and changes in isometric tension were recorded on a chart polygraph (Battaglia Rangoni, San Giorgio di Piano, Bologna, Italy). High-potassium salt solution

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(KCl) increased the tonic tension with a maximum effect obtained at 80 mM. The effect of ET-1 on the basal tone was referred to this value, taken as 100%. Relaxant–concentration curves were carried out in CC strips precontracted with 100 μM Phe (taken as 100%) and the relaxant effect induced by different drugs (SNP, Ach, IRL-1620) was referred to this value. Cumulative drug concentrations (1 nM−100 μM) were added to the bath, at different minute intervals (7 minutes for IRL-1620 and SNP and 3 minutes for Ach), to obtain a concentration-dependent curve. An in vitro pretreatment with L-NAME (100 μM) to BCN strips was performed in order to evaluate the residual NO formation in BCN penile tissue. The acute tadalafil administration was performed 30 minutes before the SNP induction.

Immunohistochemistry To analyze penile oxygenation, rats of each experimental group were intraperitoneally injected with the bioreductive drug Pimonidazole hydrochloride (Hypoxyprobe™-1, 60 mg/kg 1 hour before sacrifice), which has been recognized as a standard marker for in vivo imaging and quantification of hypoxia. Pimonidazole (hypoxyprobe) is a watersoluble substituted 2-nitrominidazole that is rapidly distributed to all tissues in the body, but it only forms adducts with proteins in hypoxic cells, that is, at an oxygen pressure equal to or lower than 10 mm Hg. Hypoxic cells are thereafter easily detected by immunohistochemistry, using a monoclonal antibody (Hypoxyprobe™-1 Mab1) that recognizes hypoxyprobe protein adducts [31– 33]. Pimonidazole as a probe for the “in vivo” detection of hypoxia has been validated previously [34]. This probe was used to verify the validity of experimental hypoxia and to evaluate a possible rescue of normoxic condition after CT treatment. Rat penis samples were rapidly removed and fixed in 4% neutral buffered formalin, dehydrated and embedded in paraffin. Immunohistochemical studies were performed on deparaffinized and rehydrated sections, according to the manufacturing instructions. Briefly, the slides were exposed to hypoxyprobe-1 Mab1 (diluted 1:50) for 40 minutes at room temperature, rinsed in PBS and 0.2% Brij 35 for seven times at 0°C and incubated with biotinylated secondary antibody (10 minutes at room temperature). Finally, the slides were incubated with streptavidin–biotin peroxidase complex (Laboratory Vision Corporation; Fremont, CA, USA) and then the reaction product was developed with the 3,3′ deamJ Sex Med 2006;3:419–431

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422 inobenzidine tetrahydrochloride (DAB) as chromogen (Sigma-Aldrich; St. Louis, MO, USA). Control were performed by processing slides lacking the primary antibody. The slides were photographed using a Nikon Microphot-FXA microscope (Nikon, Tokyo, Japan). Some sections were stained with Masson trichrome Goldener (Bio-Optica, Milan, Italy) and were used for morphological and quantitative image analysis.

HPSMC Cultures Human fetal penile smooth muscle cells were prepared from fetal male external genitalia (11– 12 weeks of gestation) obtained after spontaneous or therapeutic abortion, as previously described [20]. Cells were seeded onto 12 wells in growth medium and cultured in Petri dishes (100 mm diameter) in their growth medium. When the cultures were about 70–80% confluent, the growth medium was removed, and the cells were washed twice in PBS and incubated in phenol red- and serum-free medium containing 0.1% BSA. After 24 hours the cells were maintained in experimental hypoxic (1.5% O2, 5% CO2, and balanced N2) conditions, realized as previously described [20], in the presence or absence of tadalafil (100 nM) at different times (24 and 48 hours). Cells cultured as control were maintained for 24 and 48 hours in the same condition and in the absence of tadalafil, in normoxic atmosphere (95% air, 5% CO2). At the end of each experiment, cells were collected and further processed for RNA and protein extraction. Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Total RNA was extracted from frozen CC samples and from hPSMC using RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instructions. RNA concentration and quality were measured by spectrophotometric analysis at 260 and 280 nm. RNA integrity was assessed by electrophoresis in agarose gel. The quantitative assay was performed according to the fluorescent TaqMan method, as already reported [30]. For each sample an amount of total RNA corresponding to 400 ng was reverse-transcribed to cDNA in a final volume of 80 μL using TaqMan Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) at the following conditions: 10 minutes at 25°C; 30 minutes at 48°C, 5 minutes at 95°C. Primers and probe for β2microglobulin (chosen as reference gene), ETB, J Sex Med 2006;3:419–431

Vignozzi et al. PDE5, Enos, and nNOS mRNAs, used for quantification in samples from rat CC, and primers and probe for human PDE5 mRNA, used for the analysis in hPSMC, were all “gene expression products,” purchased from Applied Biosystems. Polymerase chain reaction (PCR) mixture (25 μL final volume) consisted of 1× final concentration of AoD mix, 1× final concentration of Universal PCR Master Mix (Applied Biosystems), and 25 ng of cDNA. Amplification and detection were performed with the ABI Prism 7700 Sequence Detection System (Applied Biosystem) with the following thermal cycler conditions: 2 minutes at 50°C, 10 minutes at 95°C, and 40 cycles at 95°C for 30 seconds and 60°C for 1 minute. Each measurement was carried out in duplicate. The mRNA quantification for rat CC samples was based on the comparative Ct method according to the manufacturer’s instructions (Applied Biosystems), as previously described [30]. The PDE5 mRNA quantification for hPSMC was performed according to the absolute method by using an external standard curve of known PDE5 mRNA starting concentrations, ranging from 3.5 × 106 to 3.5 × 10 copies/μg total RNA, to extrapolate values for unknown samples, as already published [35].

SDS-PAGE and Western Blot Analysis As previously described [35], frozen samples were directly suspended in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.25% NP-40, 1 mM Na3VO4, 1 mM phenylmethanesulfonyl fluoride [PMSF]) and homogenized for protein analysis. Thirty micrograms of protein extracts were separated on 10% denaturing SDS-polyacrylamide gel. After protein transfer to nitrocellulose membranes, the ETB, eNOS, nNOS, and PDE5 proteins were revealed by anti-ETB (1:100), antieNOS (1:200), anti-nNOS (1:1,000), and previously characterized anti-PDE5 (1:1,000) primary antibodies followed by peroxidase-conjugated IgG (1:3,000) [35,36]. Statistical Analysis Results are expressed as mean ± SEM. Statistical analysis was performed with one-way ANOVA test followed by Student’s t-test when appropriate. P < 0.05 was taken as significant. Values of halfmaximal response effective dose (ED50) and halfmaximal response inhibitory concentration (IC50) as well as potency ratio and maximal effect (Emax) were calculated by using the ALLFIT program [37].

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Ethical Committee Approval All the animal experiments were performed in accordance to the Italian Ministerial Law #116/92 and approved by the Institutional Animal Care and Use Committee of the University of Florence, Florence, Italy.

Results

Figure 1 shows the morphological effect of longterm neurotomy on penile oxygenation. In shamoperated rats only scanty cells reacted with the hypoxyprobe pimonidazole (Figure 1a). Conversely, positive hypoxyprobe staining dramatically increased in penile section of BCN rats (Figure 1b). Hypoxia was detected in the endothelial and muscular compartments of cavernous spaces. CT dosing almost completely restored penile oxygenation, as determined by the virtual absence of hypoxyprobe labeling in smooth muscle cells. Only some endothelial cells resulted positive (Figure 1c). Computer-assisted image analysis indicated that neurotomy induced a more than threefold increase in the number of hypoxic cells (P < 0.01 vs. sham) and that CT administration completely abolished it (Figure 1d). The degree of cavernous fibrosis was evaluated by Masson trichrome staining of penile specimens from the three experimental groups. Results from a typical experiment are reported in Figure 2a–c. Computer-assisted histomorphometric analysis

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Figure 1 Immunohistochemical staining of hypoxyprobe adducts in rat penis. Hypoxyprobe adducts were revealed in hypoxic cells (PO2 < 10 mm Hg) by a monoclonal antibody (magnification 10×). (a) Sham-operated rats: only scanty positive labeling is present. (b) BCN rats: massive hypoxia is present in both the endothelial (black arrows) and stromal (black head arrows) compartments. (c) BCN + CT rats: hypoxyprobe labeling is dramatically decreased; only few endothelial (black arrows) and stromal (black-head arrows) cells are positive. (d) Computer-assisted quantitative image analysis of three independent experiments. Open bars: sham; closed bars: BCN; hatched bars: BCN + CT. Sham-operated optical density was taken as 100%. *P < 0.01 vs. sham-operated.

indicated that the muscle/fiber ratio was 1.01 ± 0.02 in sham-operated rats (Figure 2d). BCN significantly decreased this ratio (0.74 ± 0.02, P < 0.01 vs. control). Muscle/fiber ratio was substantially (0.9 ± 0.02, P < 0.01 vs. BCN), but not completely (P < 0.01 vs. sham-operated), rescued by CT exposure. Table 1 reports the tension generated by rat CC strips from the different experimental groups, after exposure to 100 μM Phe or 80 mM KCl. As shown, BCN, with or without CT dosing, did not affect maximal contractile activity of CC strips. In contrast, as shown in Figure 3a, responsiveness to ET-1 resulted greatly affected. In fact, in BCN rats, the contractile response to ET-1 resulted severely depressed at all the ET-1 concentrations tested. CT supplementation completely rescued ET-1 sensitivity. This finding suggested that BCN-induced hypoxia might have changed the relative pattern of ET-1 receptor distribution, by increasing the density of the vasorelaxant ETB receptors. We therefore tested the sensitivity of rat penile strips to the selective ETB agonist IRL1620. Results are shown in Figure 3b. In shamoperated rats, IRL-1620 induced a relaxation, with an IC50 = 87.4 ± 24.5 nM, which resulted, however, of negligible entity (Emax = 15.7 ± 0.3%). BCN dramatically enhanced (P < 0.0001 vs. sham) such a relaxation. In fact, in this condition, IRL1620 almost completely relaxed CC strips, without any sensible change in the relative IC50. The relaxing activity of IRL-1620 in hypoxic strips was due

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* Figure 2 Masson trichrome staining of rat penile sections. Smooth muscle fibers are stained in red, and collagen fibers are stained in blue (magnification 4×). (a) Sham-operated rats: smooth muscle cells are present in the cavernous fibers around the sinusoids (black-head arrows) and beneath the albuginea layer (black arrows). (b) BCN rats: a clear increase in collagen fibers is present in the intersinusoidal and subtunical spaces (asterisks). (c) BCN + CT rats: smooth muscle loss is essentially prevented and fibrosis limited. (d) Computer-assisted quantitative image analysis of three independent experiments. Open bars: sham; closed bars: BCN; hatched bars: BCN + CT. *P < 0.01 vs. sham-operated, **P < 0.01 vs. BCN.

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whether BCN altered ETB receptor expression, we measured ETB at both gene (Figure 4a) and protein (Figure 4b) levels in penile homogenates from the three experimental groups. According to functional data, we found that ETB expression was significantly increased at both mRNA and protein levels (P < 0.05) by BCN, and completely restored by CT treatment.

to formation of NO, because blocking nitric oxide synthase (NOS) with L-NAME (100 μM) almost completely blunted IRL-1620 effect. In BCN rats, CT administration substantially decreased IRL1620-induced relaxation (Emax = 35.54 ± 0.95%, P < 0.0001 vs. BCN), neither completely restoring Emax to the control level (P < 0.0001), nor significantly changing the IC50. To further verify (a)

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Figure 3 Effect of bilateral cavernous neurotomy (BCN) and chronic administration of tadalafil (CT) on responsiveness to ET-1 and to the specific agonist of ETB receptor, IRL-1620, in rat CC strips. (a) Effect of increasing concentrations of ET1 (10–300 nM) on the basal tone in sham-operated (open columns), BCN (closed columns), and BCN + CT (hatched columns) CC strips. Histograms show the mean values of at least six samples for each experimental group; verticals bars indicate SEM values; *P < 0.05 vs. sham-operated, **P < 0.01 vs. sham-operated, ***P < 0.01 vs. neurotomy. (b) Effect of increasing concentrations of IRL-1620 (1 nM−100 μM) in sham-operated (closed squares), BCN (closed circle), and BCN + CT (closed triangles) Phe-precontracted CC strips. A subset of BCN strips were in vitro pretreated with L-NAME (100 μM, open circle). Ordinate: contractile tone induced by Phe (100 μM), taken as 100%. The effect of the relaxant agent was evaluated as percentage of this response. Abscissa: concentrations of IRL-1620 (molar). Each point represents mean ± SEM of at least eight samples for each experimental group.

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KCl-induced tension Mean ± SEM

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470.4 ± 161.1 405.0 ± 89.6 427.2 ± 118.9

198.9 ± 12.1 209.1 ± 13.6 241.7 ± 18.9

Tension (mg) developed by CC strips in the different experimental conditions. N = number of preparations tested. Differences were not statistically significant.

Figure 5 shows that also the sensitivity to the NO donor SNP resulted greatly affected by BCN. In fact, SNP almost completely relaxed CC strips in sham-operated rats, with an IC50 = 1.6 ± 0.7 μM. Conversely, in BCN rats, SNP IC50 resulted more than fivefold shifted to the left (IC50 = 0.28 ± 0.06 μM, IC50 ratio = 5.8 ± 2.8, P < 0.02). CT administration in BCN rats significantly enhanced SNP IC50 (IC50 = 8.3 ± 3.5 μM, P < 0.0001 vs. BCN), which resulted even higher than in sham-operated rats (P < 0.05 vs. sham). Results are shown in Figure 5a. To test the hypothesis that the changing sensitivity to SNP was due to variation in NO degradation, we repeated the in vitro experiments in the presence of 100 nM tadalafil in the organ bath. In shamoperated rats, blocking PDE5 with tadalafil (100 nM) significantly increased in vitro SNP sensitivity (SNP IC50 = 4 ± 1.2 μM; SNP + tadalafil IC50 = 0.023 ± 0.005 μM, IC50 ratio = 173 ± 65, P < 0.0001, Figure 5b). Conversely, acute incubation with tadalafil (100 nM) did not affect SNP responsiveness in BCN rats. In fact, in this experimental condition, the two sigmoidal relationships can be fitted by the same equation (P = 0.414, Figure 5c). CT administration in BCN rats almost fully restored sensitivity to acute tadalafil exposure (IC50 ratio = 65 ± 38, P < 0.001, Figure 5d). To verify whether BCN might affect PDE5 expression, we measured it. Results are reported in Figure 6. As shown, BCN significantly reduced PDE5 gene (P < 0.01 vs. sham, Figure 6a) and protein (P < 0.05 vs. sham, Figure 6b) expression, while CT rescued it. To further investigate whether changing PDE5 gene expression was directly related to the hypoxic environment, we performed experiments in isolated hPSMC, exposed to hypoxia according to a previously described protocol [20]. We observed (Figure 7) that, even after 24-hour hypoxia (1.5% O2, Figure 7a), there was a significant (P < 0.05) decrease in PDE5 gene expression, which was

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maintained after 48 hours (Figure 7b) and not rescued with a simultaneous incubation with tadalafil (100 nM). Similar results were obtained after 48 hours, analyzing protein expression by Western blot (not shown). Figure 8 shows the effect of BCN on Ach responsiveness in CC strips. In sham-operated rats, Ach dose-dependently relaxed Phe-precontracted strips (IC50 = 0.47 ± 0.18 μM; Emax = 41.5 ± 2%). BCN significantly reduced Ach Emax

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Figure 4 Effect of bilateral cavernous neurotomy (BCN) and chronic administration of tadalafil (CT) on ETB expression in rat CC. (a) Quantitative detection of ETB mRNA by real-time RT-PCR. Data are expressed as mean ± SEM of arbitrary units (a.U.) calculated according to the comparative Ct method and using the β2-microglobulin housekeeping gene for normalization. White column: sham-operated (N = 16); black column: BCN (N = 19); hatched column: BCN + CT treatment (N = 15). (b) Western blot analysis of ETB protein in CC from experimental rats. Upper panel: densitometric evaluation of ETB protein expression, calculated as percentage of band intensity over the shamoperated sample taken as 100%. Data are mean ± SEM. White column: sham-operated (N = 4); black column: BCN (N = 4); hatched column: BCN + CT treatment (N = 4). Lower panel: a representative Western blot. Molecular weight (Mw) marker (kDa) is indicated to the right of the blot. *P < 0.05 vs. any other groups.

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Figure 5 Effect of bilateral cavernous neurotomy (BCN) and chronic administration of tadalafil (CT) on NO sensitivity in rat CC strips precontracted with Phe. (a) Effect of increasing concentrations of SNP (1 nM−100 μM) in CC strips from shamoperated (closed circles), BCN (closed squares), and BCN + CT (closed triangles) rats. Each point represents mean ± SEM of at least eight samples for each experimental group. (b–d) Effect of acute incubation with tadalafil (100 nM) on responsiveness to SNP in rat CC strips from sham-operated (b), BCN (c), or BCN + CT (d) rats. Each point represents mean ± SEM of at least six samples for each experimental group. Ordinate: contractile tone induced by Phe (100 μM), taken as 100%. The effect of the relaxant agents was evaluated as percentage of this response. Abscissa: increasing concentrations of SNP (molar).

(Emax = 25.74 ± 0.74, P < 0.0001 vs. sham). However, in this case, CT treatment did not rescue Ach responsiveness (Emax = 20.33 ± 0.5, P < 0.0001 vs. sham). No variation in Ach IC50 was observed among groups. Blocking NO formation with LNAME (100 μM) further reduced Ach-induced relaxation, even in the hypoxic strips. Because Ach-induced relaxation is dependent on NO formation, we investigated the expression of the two main isoenzymes of NOS, that is, eNOS and nNOS, at both gene (Figure 9a,c) and protein (Figure 9b,d) levels. Relative expression of both isoenzymes resulted significantly depressed, although not completely abrogated, by BCN (eNOS gene P < 0.05; protein P < 0.001 vs. sham; J Sex Med 2006;3:419–431

nNOS gene and protein both P < 0.05 vs. sham). CT dosing did not rescue BCN-induced NOS hypoexpression (eNOS gene P < 0.05; protein P < 0.001 vs. sham; nNOS gene P < 0.05 and protein P < 0.01 vs. sham, all not significant vs. BCN). Discussion

This study demonstrates, for the first time, that, in an experimental model of RRP-induced erectile dysfunction (BCN), CT treatment restores several of the investigated neurotomy-induced penile alterations, including PDE5 expression and in vitro responsiveness to tadalafil. This is probably due to an increased cavernous oxygenation, which

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induced penile hypoxia is coupled to severe alterations of muscular/fiber ratio [13,14]. CT treatment almost completely restores penile oxygenation and muscular/fiber ratio at histology. Molecular mechanisms underlying this effect were only partially clarified by the present study. We previously reported that in hPSMC cultures [20] and in penile rabbit explants [23] one of the main counter-regulatory responses to penile hypoxia is the overexpression of the ET-1 receptor subtype ETB, while ETA subtype resulted substantially unchanged. The physiological meaning of this hypoxia-induced ETB upregulation in penile cavernous tissue is still obscure, but it is important to note that ETB signaling is positively coupled to formation of several vasodilator effec-

4

* *

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180

**

150 120

*

90 60 (a)

30 Mw (kDa)

0

115 PDE5 (98 kDa) 82 Sham

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Figure 6 Effect of bilateral cavernous neurotomy (BCN) and chronic administration of tadalafil (CT) on PDE5 expression in rat CC. (a) Quantitative detection of PDE5 mRNA in CC from experimental rats by real-time RT-PCR. Data are expressed as mean ± SEM of arbitrary units (a.U.) calculated according to the comparative Ct method and using the β2-microglobulin housekeeping gene for normalization. White column: sham-operated (N = 9); black column: BCN (N = 8); hatched column: BCN + CT (N = 8). (b) Western blot analysis of PDE5 protein in CC from experimental rats. Upper panel: densitometric evaluation of PDE5 protein expression calculated as percentage of band intensity over the sham-operated sample taken as 100%. Data are mean ± SEM. White column: sham-operated (N = 4); black column: BCN (N = 4); hatched column: BCN + CT (N = 4). Lower panel: a representative Western blot. Molecular weight (Mw) markers (kDa) are indicated to the right of the blot. *P < 0.05, **P < 0.01 vs. BCN.

preserves penile smooth muscle content and function. In this study we demonstrate that BCN induces a severe penile hypoxia. In fact, hypoxyprobe labeling, a generally accepted probe of hypooxygenation [31–33], was dramatically increased after a 3-month interval from surgical neurotomy. Our finding directly confirms previous hypotheses on the neurotomy-induced penile hypoxia [12– 16]. In addition, we confirm that neurotomy-

24 hours

120 100

PDE5 mRNA % over normoxia

% PDE5 positivity over control

(b)

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40

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PDE5 mRNA % over normoxia

PDE5/β2-microglobulin mRNA (a.U.)

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80 *

*

60 40 20 0

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Figure 7 Effect of hypoxia (1.5% O2) on PDE5 mRNA expression in hPSMC in culture. Quantitative detection of PDE5 mRNA was performed by real-time RT-PCR. mRNA quantification was carried out by absolute method, using a standard curve of known PDE5 mRNA concentrations to extrapolate values for unknown samples. In normoxic condition (white column), the basal expression of PDE5 was 2 ± 0.9 and 1.9 ± 0.6 × 107 molecules/μg at 24 hours (a) and 48 hours (b), respectively (N = 3). Data in hypoxic conditions, with (hatched column) or without (black column) tadalafil (100 nM) incubation, are expressed as percentage over normoxia, taken as 100%. Data are mean ± SEM. *P < 0.05 vs. normoxia.

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Response (%)

100 90 80 70 60 50

sham BCN BCN + CT BCN + L-NAME

10–9

10–8

10–7 Ach (M)

10–6

10–5

10–4

Figure 8 Effect of bilateral cavernous neurotomy (BCN) and chronic administration of tadalafil (CT) on responsiveness to acetylcholine (Ach) in rat CC precontracted by phenylephrine (Phe). Effect of increasing concentrations of Ach (1 nM−100 μM) is shown in sham-operated (closed squares), BCN (closed circles), and BCN + CT (closed triangles) CC strips. A subset of BCN strips were in vitro pretreated with L-NAME (100 μM, open circle). Ordinate: contractile tone induced by Phe (100 μM), taken as 100%. The effect of Ach was evaluated as percentage of this response. Abscissa: increasing concentrations of Ach (molar). Each point represents mean ± SEM of at least 12 samples for each experimental group.

tors, such as prostanoids [38], epoxygenase metabolites [39], and NO [23]. Hence, in hypoxic penis, ETB upregulation may represent a compensatory mechanism to buffer the vasoconstrictor activity of ET-1 and therefore to limit hypoxia. We now report similar results in the rat penis. In fact, BCN-induced chronic hypoxia stimulates an increase of both ETB sensitivity and expression. Accordingly, in BCN rats, responsiveness to ET1 was decreased, while sensitivity to IRL-1620 resulted increased, along with an increase in ETB receptor gene and protein expression. Interestingly, restoring penile oxygenation through CT administration completely reverted ETB overexpression and reduced hypersensitivity to IRL-1620. A similar compensatory change may be hypothesized for the BCN-induced PDE5 downregulation. PDE5 is an intracellular enzyme highly expressed in human [35] and rat [30] penis, which hydrolyzes cyclic guanosine monophosphate (cGMP) to inactive metabolites. Accumulation of cGMP is the main prerequisite for penile erection and oxygenation. PDE5 inhibition, by means of chemical compounds (as sildenafil, tadalafil, and vardenafil) or gene silencing [40], is, indeed, the main strategy to increase penile NO sensitivity, cGMP accumulation, and erections. We now J Sex Med 2006;3:419–431

found that hypoxic penile tissue or cells have a natural silencing of PDE5 gene and protein expression, most probably aimed to increase the NO-cGMP vasodilator pathway. In fact, the sensitivity to a NO donor, as SNP, was significantly increased in hypoxic CC strips. Penile expression of PDE5 is the cornerstone for PDE5i proerectile activity [41]. Resistance to tadalafil activity, hypersensitivity to SNP, and reduced PDE5 expression were all reverted by CT dosing, most probably because penile oxygenation was restored. Hypoxia-induced PDE5 downregulation was also observed in a previously described model of isolated hPSMC [20,23], which express elevated concentrations of PDE5 mRNA (2 × 107 molecules/ μg total RNA), according to their penile origin [35]. In apparent contrast to the in vivo results, in hPSMC incubation with tadalafil (100 nM) at different times did not rescued the hypoxia-induced downregulation of PDE5 expression. Hence, we speculate that it was the CT-induced penile oxygenation, and not tadalafil itself, that rescued PDE5. Our findings may have clinical correlates. In fact, one of the reasons for PDE5i resistance in patients who underwent RRP may be ascribed to the hypoxia-induced penile damages, including downregulation of PDE5 expression. In these patients, in vivo CT dosing not only may improve response to PDE5i, but also may limit penile fibrosis of the smooth muscle compartment. However, CT administration was unable to significantly improve NO formation, as tested by Ach responsiveness, which, as previously reported [18], resulted depressed by hypoxia. This might be due to a persistent alteration in the expression of the key enzymes responsible for NO synthesis, as nNOS and eNOS, even after CT dosing. It should be interesting to evaluate whether in less severe models of neuronal injury, as monolateral cavernous neurotomy, CT might improve also these other aspects of penile function. It is important to note that in hypoxic penile tissues NO synthesis, although reduced, is still present because its inhibition with L-NAME substantially hampered IRL-1620 and Ach effects, which are know to be mediated by NO formation [18,23]. In conclusion, our hypothesis is that CT by decreasing PDE5 activity—and therefore cGMP degradation—amplifies NO signaling, which resulted reduced, but not abolished, by neurotomy. Amplifying a depressed NO signaling might help to improve tissue oxygenation, which, in turn, in one hand would limit fibrosis, and in the other

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(c)

eNOS/β2-microglobulin mRNA (a.U.)

(a) 8 6 *

4

*

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8 6 *

*

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% nNOS positivity over control

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120 100 80 60 40 20 0

90 *

60

**

30 Mw (kDa) 182

0

nNOS (155 kDa)

115 Sham

BCN BCN + CT

Figure 9 Effect of bilateral cavernous neurotomy (BCN) and chronic administration of tadalafil (CT) on NOS expression in rat CC. (a,c) Quantitative detection by real-time RT-PCR of mRNA for eNOS (a) and nNOS (c) in the different experimental groups. Data are expressed as mean ± SEM of arbitrary units (a.U.) calculated according to the comparative Ct method and using the β2-microglobulin housekeeping gene for normalization. White column, sham-operated (N = 9); black column: BCN (N = 7); hatched column: BCN + CT (N = 8). (b,d) Western blot analysis of eNOS (b) and nNOS (d) protein in CC from experimental rats. Upper panels: densitometric evaluation of protein expression calculated as percentage of band intensity over the sham-operated sample taken as 100%. Data are mean ± SEM. White column: sham-operated (eNOS, N = 5; nNOS, N = 4); black column: neurotomy (eNOS, N = 7; nNOS, N = 5); hatched column: neurotomy plus CT treatment (eNOS, N = 7; nNOS, N = 5). Lower panels: a representative Western blot. Molecular weight (Mw) markers (kDa) are indicated to the right of the blot. *P < 0.05, **P < 0.001, ***P < 0.001 vs. sham-operated.

one would preserve PDE5 expression and potential PDE5i responsiveness. This hypothesis might explain previously reported success with other vasodilators as prostanoids [42,43]. However, one major limitation of this study is that it is conducted only ex vivo (evaluation of hypoxia) or in vitro, and therefore present findings should be confirmed in other models. In particular, it must be verified whether CT in neurectomized animals allows restoring erectile function also in vivo. Corresponding Author: Mario Maggi, MD, Andrology Unit, Department of Clinical Physiopathology, University of Florence, V.le G. Pieraccini, 6, 50139 Florence, Italy. Tel: (+39) 0554271415; Fax: (+39) 0554271413; E-mail: [email protected]fi.it Conflict of Interest: None declared.

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