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Urotensin II: A Novel Target in Human Corpus Cavernosum
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Roberta d’Emmanuele di Villa Bianca, PhD,*† Giuseppe Cirino, PhD, FBPharmacolS,*† Emma Mitidieri, PhD,† Ciro Coletta, PhD,† Gianluca Grassia, PhD,† Fiorentina Roviezzo, PhD,† Paolo Grieco, PhD,‡ Ettore Novellino, PhD,‡ Ciro Imbimbo, MD,* Vincenzo Mirone, MD,* and Raffaella Sorrentino, PhD*† *University of Naples, Federico II—Interdepartmental Research Centre for Sexual Medicine (CIRMS), Naples, Italy; † University of Naples Federico II—Department of Experimental Pharmacology, Naples, Italy; ‡University of Naples Federico II—Department of Pharmaceutical and Toxicological Chemistry, Naples, Italy DOI: 10.1111/j.1743-6109.2009.01450.x
ABSTRACT
Introduction. Urotensin II (U-II) is a cyclic peptide originally isolated from the teleost neurosecretory system and subsequently identified in other species, including man. U-II was identified as the natural ligand of an orphan G-protein coupled receptor (UT receptor). U-II and UT receptor are expressed in a variety of peripheral organs and especially in cardiovascular tissue. U-II caused both constrictor and vasodilator effect, depending by vascular bed. The in vivo functional consequences of U-II on the cardiovascular hemodynamics are not clearly understood. Aim. To investigate the presence of UT receptor and the effect of U-II in human corpus cavernosum (HCC) strips. To evaluate the effect of U-II in vivo in anesthetized rats. Methods. UT receptor expression as protein and as mRNA were assessed by Western blot and reverse transcriptase polymerase chain reaction. Next, the UT receptor localization was evaluated by immunohystochemical analysis. By using HCC strips, with or without endothelium, the effect of U-II (0.1 nM–10 mM) was evaluated. In order to asses the nitric oxide (NO) involvement, the strips were incubated with N (G)-nitro-L-arginine methyl ester (NO synthase inhibitor, 100 mM). U-II (0.1, 0.3, 1.0 nmol/rat) effect in vivo was studied in anesthetized rats by monitoring the intracavernous and systemic blood pressure. Main Outcome Measures. HCC expresses the UT receptor and its activation, by UII, causes an endothelium- and NO-dependent relaxation. Results. UT receptor is expressed in human and rat corpus cavernosum. In HCC UT receptor is localized on endothelial cells. U-II significantly relaxed HCC strips in endothelium- and –NO-dependent fashion. The peptide caused a significant increase in intracavernous pressure in anesthetized rats. Conclusion. This study demonstrates that UT receptor is expressed on the endothelium of HCC. U-II/UT receptor system is involved in HCC function and it involves endothelium and NO pathway. Thus, U-II/UT receptor pathway could be involved in erectile function. d’Emmanuele di Villa Bianca R, Cirino G, Mitidieri E, Coletta C, Grassia G, Roviezzo F, Grieco P, Novellino E, Imbimbo C, Mirone V, and Sorrentino R. Urotensin II: A novel target in human corpus cavernosum. J Sex Med 2010;7:1778–1786. Key Words. Human Corpus Cavernosum; Urotensin II Receptor; Urotensin II; Nitric Oxide; Rat; Erection Physiology
Introduction
U
rotensin II (U-II), a cyclic octapeptide, was first recognized almost 30 years ago as an important teleost fish hormone [1,2]. It is now known that U-II acts as a vasoactive peptide in mammals, by binding to the orphan G-proteincoupled receptor 14 (GPR14) namely, UT recepJ Sex Med 2010;7:1778–1786
tor. U-II and UT receptor are expressed in variety of peripheral organs (liver, kidney, endocrine glands) and especially in cardiovascular tissues (cardiomyocyte, endothelium, and vascular smooth muscle cells) [2,3]. Moreover, both the peptide and the receptor have been detected in some areas of the central nervous system [3,4]. U-II is present in pmolar concentrations in blood © 2009 International Society for Sexual Medicine
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U-II Involvement in Erectile Function in healthy individuals and the U-II/UT receptor system seems to play an important role in cardiovascular function [5,6]. Initial studies have shown a vasoconstrictive action of U-II, which is more potent than endothelin 1 depending on the vessel [7]. Indeed, the vascular response to U-II varies, depending upon the species, type of blood vessel, concentration of U-II, and route of administration [7–9]. For instance, U-II contracts many rat large capacitance vessels such as thoracic aorta, carotid, pulmonary, and coronary arteries, and this vasconstrictor effect at the least on the rat thoracic aorta is mediated by phospholipase C and protein kinase C-dependent pathways [10–12]. Conversely, U-II dilates rat and human small resistance arteries [13,14]. On this regard, it has been reported that U-II produces a marked nitric oxide (NO)dependent vasodilator response in rat isolated small renal arteries as well as in other vascular beds [13–15]. The effect of U-II has been also evaluated in different cell type, in particular, U-II causes a dose-dependent increase in intracellular Ca+2 in human aortic endothelial cell [16]. U-II has been also tested in vivo experimental animals and in human with divergent results. In anesthetized monkey, systemic administration of U-II caused circulatory collapse as result of the profound vasoconstrictor effect of the peptide [3]. Conversely, infusion of U-II did not cause any significant change in heart rate, mean arterial pressure or cardiac index in healthy male volunteers [17]. In anesthetized rats intravenous injection of U-II showed an hypotensive effect partially mediated by NO [18]. Penile corpus cavernosum is an highly vascularized tissue whose function is dependent upon a balance between the vasodilatory and the vasoconstrictory tone. In penile erection there is a strong involvement of the vascular system and the L-arginine/NO pathway plays a major role [19,20]. Indeed, it is now widely accepted that erectile dysfunction (ED) is predominantly a vascular disease and ED is considered an early sign of cardiovascular disorders [21,22]. In the present study, by using human corpus cavernosum (HCC) obtained by a standardized surgical procedure [23], we have demonstrated that UT receptor is expressed in human and rat penile tissues. Functional studies, performed in vitro, showed that U-II plays a role in human tissue. Finally, the intracavernous administration of U-II to rats in vivo elicited penile erection.
Aims
To evaluate the expression of UT receptor and the physiopharmacological effect of U-II in HCC strips. Methods
Peptide The human U-II was synthesized and purified at the Department of Pharmaceutical and Toxicological Chemistry of the University of Naples, Federico II. The peptide was obtained by solid-phase peptide synthesis as previously reported [24]. Purification was achieved using a semi-preparative reversedphase high-performance liquid chromatography (HPLC) C18 bonded silica column (Vydac 218TP1010; The Separations Group Inc., Hesperia, CA, USA). The purified peptide was 99% pure as determined by analytical reversed-phase HPLC. The correct molecular weights were confirmed by mass spectrometry and amino acid analysis. Human Tissue In male-to-female transsexual surgical procedures, the penis and testicles are amputated and a neovagina is created to simulate female external genitalia. Patients undergo appropriate hormonal pre-treatment with antiandrogens and estrogens to adapt to female appearance, and the therapy is discontinued 2 months before surgery. The corpora cavernosa were carefully excised from the penis immediately after amputation and placed in ice-cold oxygenated Krebs solution [23]. All patients were informed of all procedures and gave their written consent. The protocol was approved by the Ethics Committee of the Medical School of the University of Naples Federico II. Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) The presence of GPR14 was determined by quantitative PCR. Briefly, mRNA from HCC and human colon tissue was extracted by using TRIzol reagent (GIBCO; GIBCO, Milan, Italy), according to the manufacturer’s recommendations and then cDNA was prepared [25]. Reverse transcription was performed and 100 ng of the RNA samples described earlier were used for quantitative PCR. Samples were run in triplicate in 50 mL reactions by using an ABI PRISM 7500 sequence detector system (Applied Biosystems, Foster City, CA, USA). Amplification was done using Hs01027998_s1 TaqMan Gene Expression assay J Sex Med 2010;7:1778–1786
1780 (Applied Biosystems), amplification set up and cycling were performed according to manufacturer’s recommendations.
Western Blot Analysis Human or rat corpus cavernosum were homogenized in modified RIPA buffer (Tris-HCl 50 mM, pH 7.4, Triton 1%, sodium deoxycholate 0.25%, NaCl 150 mM, ethylenediaminetetraacetic acid 1 mM, phenylmethylsulphonyl fluoride 1 mM, aprotinin 10 mg/mL, leupeptin 20 mM, NaF 1 mM, sodium orthovanadate 1 mM) by liquid nitrogen. After centrifugation of homogenates at 12,000 rpm for 15 minutes, 40 mg of denatured proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to a polyvinylidene fluoride membrane. Membranes were blocked by incubation in phosphate-buffered saline (PBS) containing 0.1% v/v Tween 20 and 5% nonfat dry milk for 2 hours, followed by overnight incubation at 4°C with rabbit polyclonal antibody for GPR14 (H-90) (1:1,000; Santa Cruz Biotechnology, Inc., Heidelberg, Germany). The filters were washed extensively in PBS containing 0.1% v/v Tween 20, before incubation for 2 hours with horseradish peroxidase-conjugate anti mouse secondary antibody (1:5,000). Membranes were then washed and developed using enhanced chemiluminescence substrate (Amersham Pharmacia Biotech, San Diego, CA, USA). Immunofluorescence HCC samples were fixed in 4% buffered formalin and paraffin-embedded. Cross-sections were cut (6 mm) and used for the detection of specific proteins by immunofluorescence. Paraffin sections, after being de-waxed and rehydrated, were boiled for 30 minutes in citrate buffer for antigen retrieval. The sections were incubated in blocking buffer (PBS with 1% casein) for 1 hour at room temperature in a humidified chamber. Endogenous biotin was blocked with the Avidin/Biotin Blocking Kit (Vector Laboratories, Burlingame, CA, USA). The sections were incubated (4°C) overnight with antiGPR14 goat polyclonal antibody (1:100; P-15, Santa Cruz Biotechnology) diluted in 1% blocking reagent (Perkin Elmer; Santa Cruz Biotechnology, Inc., Heidelberg, Germany)/0.3% Triton X-100 in PBS. Sections incubated with 1% goat serum were used as negative controls. Subsequently, sections were incubated with donkey anti-goat secondary antibody Texas Red conjugated (1:100; Jackson Immunoresearch) for 30 minutes. After washing, sections were incubated for 1 hour with mouse J Sex Med 2010;7:1778–1786
d’Emmanuele di Villa Bianca et al. monoclonal anti-a-smooth muscle actin (a-SMA) antibody fluorescein isothiocyanate (FITC) conjugated (1:250; Sigma, Milan, Italy), or for 2 hours with rabbit polyclonal anti-von Willebrand factor (vWF; 1:100; Dako) and donkey anti-rabbit secondary antibody FITC conjugated (1:100; Jackson Immunoresearch) for 30 minutes. 4′,6-Diamidino2-phenylindole was used to stain the nuclei. Images were taken with the aid of a Leica DFC340 FX video-camera (Leica, Milan, Italy) connected to a Leica DMRB microscope using Leica Application Suite software V2.4.0 and processed with Adobe Photoshop 7.0 software (Adobe Systems Inc.).
HCC Strips Longitudinal strips (2 cm) of HCC were dissected from the trabecular structure of the penis and isolated [23]. Krebs solution had the following composition (mM): 115.3 NaCl; 4.9 KCl; 1.46 CaCl2; 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, Ugo Basile, Comerio, Italy) and changes in tension were recorded continuously by using a polygraph linearcorder (WR3310, Graphtec, Yokohama, Japan). 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; 3 mM; Sigma) contractions until three equal responses were obtained. Endothelial integrity was assessed by using acetylcholine (Ach; 0.01–10 mM; Sigma). Strips without a functional endothelium (Ach responsive) were obtained by incubating in distilled water for 15 seconds. A concentrationresponse curve to U-II (0.1 nM–10 mM) was obtained in the presence or absence of endothelium, using HCC strips pre-contracted with PE (3 mM). To assess the involvement of NO, we incubated the strips for 30 minutes with N (G)-nitroL-arginine methyl ester (L-NAME; 100 mM, Sigma), NOS inhibitors, before U-II challenge. In another set of experiments a concentrationresponse curve to U-II (0.1 nM to 10 mM) was obtained using HCC strips under resting condition. Data were calculated as % of relaxation to PE tone and expressed as the mean ⫾ standard error of the mean (SEM) from eight separate specimens from four different tissues. The results were analyzed by using analysis of variance (anova) followed by Bonferroni post hoc test.
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Monitoring Intracavernous Pressure (ICP) in Anesthetized Rats The present study was performed in accordance with the guidelines of Italian law (N. 116/1992) and European Council law (N.86/609/CEE) for animal care. Male Wistar rats (200–250 g) were used (Harlan, Udine, Italy). The experimental procedures were approved by the Animal Ethics Committee of the University of Naples. Animals were kept under laboratory conditions (temperature 23 ⫾ 2°C, humidity range 40–70%, 12-hour light/dark cycle). Food and water were fed ad libitum. Rats were anesthetized with an intraperitoneal injection of urethane (1 g/kg), so that the rats breathed spontaneously during the experiment. For continuous systemic blood pressure measurements, an heparinized (5 I.U./mL) polyethylene catheter was introduced into the carotid artery connected to a pressure transducer (BLPR-2, 2Biological Instruments, Milan, Italy). With a midline perineal incision, followed by blunt dissection of the overlying striated muscles, entrance to the tunica albuginea of the crus corpus cavernosum was achieved. A 26G-gauge needle attached to an heparinized (10 I.U./mL) polyethylene catheter was inserted into the crus corpus cavernosum and the ICP was monitored with a pressure transducer (BLPR-2, 2Biological Instruments, Milano, Italy). These parameters were recorded and data acquisition and calculations were performed using a computer system (Biopac, 2Biological Instruments, Milan, Italy). For pharmacological evaluation via the intracavernous route, a 26G-gauge needle was placed at the other crus for drug injection. U-II was given at doses of 0.1, 0.3, 1.0 nmol/rat dissolved in 50 mL saline. To validate the experimental model we used Ach (2.5, 5.0, 25.0 nmol/rat). Saline served as the control vehicle. Data were calculated as well as delta (mm Hg) of ICP and area under the curve (mm Hg ¥ min) and expressed as mean ⫾ SEM from five separate experiments. The changes in systemic blood pressure were calculated as differences from basal values following intra-cavernous drug injection (delta mm Hg) and expressed as mean ⫾ SEM. Data were analyzed using anova followed by Bonferroni. Main Outcome Measures UT receptor is expressed in HCC and is localized in endothelial cells. U-II causes an endotheliumand NO-dependent relaxation in HCC strips. Intracavernous administration of U-II in vivo to
Figure 1 Urotensin II receptor (GPR14) is expressed in human and rat corpus cavernosum. (A) Representative western blot for urotensin II receptor (GPR14) in human (HCC) and rat corpus cavernosum. b-actin was used as loading control. (B) Quantitative reverse transcriptase polymerase chain reaction for urotensin II receptor (GPR14) in human corpus cavernosum and human colon. The data represent the mean ⫾ standard error of the mean of three different human and rat specimens.
rats causes an increase in ICP. U-II/UT receptor pathway is involved in erectile function. Results
Tissue Distribution of U-II Receptor Western blot analysis showed that UT receptor is expressed in both human and rat corpus cavernosum tissue (Figure 1A). In addition, the quantitative RT-PCR demonstrated the presence of UT receptor mRNA in HCC tissue, as control we used human colon tissue (Figure 1B). The immunofluorescence analysis performed to locate UT receptor expression demonstrated a robust signal for UT receptor in HCC (Figure 2B, C). Negative control slides showed no signal (Figure 2A). The colocalization of the UT receptor with endothelial cell marker, vWF, clearly showed a strictly overlapping of the signals indicating that the UT receptor was expressed by endothelial cells of the vessels (Figure 2B). Instead, only some cells in the media of the vessels showed immunoreactivity for UT receptor (Figure 2C). A signal for the UT receptor, even if less evident, was also visible in the endothelium of sinusoids (data not shown). Effect of U-II in HCC Strips U-II (0.1 nM–10 mM) administration in HCC strips in the absence of endothelium, under resting J Sex Med 2010;7:1778–1786
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Figure 2 Localization of the urotensin II receptor (GPR14) in human corpus cavernosum. Photomicrographs show cellular localization of the GPR14 protein (red) with immunofluorescence. (A) negative control. Panel B and C: Sections were doubled stained with an endothelial cell marker (von Willebrand factor [vWF]; green) (B), or with a smooth muscle cell marker (a-SMA; green) (A, C). 4i,6-Diamidino-2-phenylindole (blue) was used to stain the nuclei. The merges showed the GPR14 colocalization (yellow). Results illustrated are from a single experiment and are representative of three different specimens.
condition, did not cause any vasoconstrictor effect (data not shown). In the presence of stable tone elicited by PE (3 mM), U-II (0.1 nM–10 mM), caused a concentration- and endotheliumdependent relaxation (Figure 3A; ***P < 0.001). The pEC50 of U-II was 8.291 ⫾ 0.453 (half maximal effective concentration [EC50] 5.1 nM). Endothelial removal abrogated the response to U-II (Figure 3A). In order to assess further the potential involvement of NO in the U-II-induced relaxation response, the strips were incubated with L-NAME, a NOS inhibitor (100 mM) for 30 minutes. L-NAME abolished U-II-induced relaxation (Figure 3B; **P < 0.01).
U-II Increases Intracavernosal Pressure in Anesthetized Rats In order to evaluate whether U-II causes penile erection in vivo, we monitored the ICP after administration of the peptide into the rat corpus J Sex Med 2010;7:1778–1786
cavernosum. Administration of U-II (0.1; 0.3; 1.0 nmol/rat) induced a dose-dependent increase in ICP, thus penile erection, as shown in Figure 4A, B. The data are expressed as delta of increase in ICP (Figure 4A, ***P < 0.001) as well as area under the curve (Figure 4B, ***P < 0.001). To validate the experimental procedure we used Ach (2.5, 5.0, 25.0 nmol/rat), which caused a dosedependent increase in ICP (Figure 4C; *P < 0.05, **P < 0.01). Injection of saline (vehicle; 50 mL) caused no appreciable effect (Figure 4). In addition, the peptide administration did not modify significantly the mean arterial blood pressure at all doses tested (Figure 4D). Conversely, Ach reduced the blood pressure in a dose-dependent manner (data not shown). Discussion
U-II, that was originally identified as one of a number of peptide hormones secreted by the fish
U-II Involvement in Erectile Function
Figure 3 Urotensin II (U-II) effect on human corpus cavernosum (HCC) strips. (A) U-II relaxed HCC strips precontracted with PE (3 mM) in presence of endothelium (***P < 0.001). (B) N (G)-nitro-L-arginine methyl ester (L-NAME; 100 mM) abolished U-II-induced relaxation (***P < 0.001). Data expressed as mean ⫾ standard error of the mean (N = 8) were analyzed by analysis of variance and by Bonferroni as post-test. CTR = control.
1783 caudal neurosecretory system has been recently shown to be a very potent vasoactive peptide in mammalian vessels [1,3,26,27]. The peptide and its receptor are abundantly distributed in human heart, brain, pancreas, skeletal muscle, vascular smooth muscle and endothelial cells, spinal cord, and endocrine tissue [3,28]. This wide distribution suggests that U-II may serve as circulating hormone to participate in the regulation of many physiological processes. The vasoactive effects of U-II seem to be dependent upon the caliber of the vessels and the species [9–11]. Initially, U-II was indicated as the most potent vasoconstrictor agents, specially, in rat thoracic aorta, where neither removal of endothelium, nor inhibition of NO synthesis, affected the peptide-induced contraction [3,29]. Conversely, it has been suggested that U-II-effect in resistance vessels in rat and human, causes vasodilation involving NO pathway [13–15]. Thus, the cardiovascular actions of this peptide can be influenced by regional factors such as receptor distribution and the species considered. To date it is unclear whether the predominant action of U-II in human disease will be deleterious or protective. In fact it has been reported that U-II has (transient) coronary vasodilator and vasodepressor (hypotensive) effects on SHR and an anti-apoptotic effect on human
Figure 4 Effect of urotensin II (U-II) on intracavernous and systemic pressure in anesthetized rats. U-II (0.1, 0.1, 1 nmol/rat) caused significant dosedependent increases in intracavernous pressure (ICP) compared with saline. The data are expressed in (A), as delta in mm Hg (***P < 0.001; N = 5) and in (B), as area under the curve (***P < 0.001; N = 5). (C) Ach (2.5, 5, 25 nmol/rat) caused a dosedependent increase in intracavernous pressure (*P < 0.05, **P < 0.01, N = 5). (D) U-II did not cause any change in arterial blood pressure.
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1784 endothelial cells [30]. Recent findings suggest that the endothelium may be a key determinant for changes in vasomotor tone to U-II in vivo. In fact an alteration of U-II/UT receptor pathway contributes to the development of several cardiovascular disorders associated to endothelial dysfunction. Indeed, elevated plasma levels of U-II have been detected in patients with various cardiovascular disease states including heart failure, hypertension, renal dysfunction, and diabetes [31–34]. In penile erection there is a strong involvement of the vascular system and the L-arginine/NO pathway plays a major role [19,20]. Following the finding that there is a possible link between U-II and NO [13,14] here we have addressed the question of whether U-II could act as an endogenous mediator in HCC and thus be involved in human erectile function. In this regard, a previous study has indicated that a central administration of U-II had no effect on apomorphine-induced penile erection in mice [35]. However, there are no published studies using human penile tissue. In order to address this issue, we used HCC obtained from patients undergoing sex change, which we have already shown to be a reliable tissue source to study receptors and mediators involved in human physiology [23,25,36]. At the present there are no data available in the literature on a possible influence of hormone therapy and UT receptor in human. First, we addressed the question if the human tissue expresses the receptor and where it is localized. The human tissue possesses the UT receptor as protein as well as mRNA. Interestingly, endothelial cells of the HCC tissue strongly stained for UT receptor implying that U-II might play a role in the HCC vasodilatory response. These data, taken together with the finding that U-II is present in picomolar concentrations plasma of healthy individuals [5] and that the U-II/UT receptor system has been involved in cardiovascular system, strongly suggested a functional role for U-II/UT receptor pathway in corpus cavernosum function. In our experimental condition there was no evidence of immunoreactivity for U-II either in vessel or in the lacuna endothelial line of human tissues (data not shown). The functional studies performed on HCC strips showed that U-II did not have a vasoconstrictor effect on human tissue. These data could be explained by the finding that the UT receptor is mainly localized on the endothelium of HCC, as demonstrated by immunofluorescence studies. Conversely, HCC strips pre-contracted with PE relaxed to U-II in a J Sex Med 2010;7:1778–1786
d’Emmanuele di Villa Bianca et al. concentration-dependent manner. In our experimental condition we used nmolar concentration in apparent contrast with plasma level reported in the literature (pmolar). This discrepancy could be ascribed to a different bioavailability UII in vitro, e.g., in the organ bath. In other words, the peptide in our experimental condition is not readily available to the tissue as in the in vivo situation when it circulates; in addition, in vitro, most likely there are some unspecific bindings. The endothelium removal abrogated U-II vasorelaxant effect, suggesting a link with the NO pathway in this tissue. To test this hypothesis we incubated HCC strips with L-NAME, prior to addition of U-II. The inhibition of Larginine/NO pathway abrogated U-II-induced relaxation with a profile overlapping the inhibition operated by the removal of endothelium supporting our working hypothesis. Our results are consistent with several evidence indicating that stimulation of UT receptors on animal vascular endothelial cells can trigger the release of NO [13,15,37–40]. In addition, U-II binding sites have been localized to vascular endothelial cells, confirming that U-II stimulates NO-dependent vascular relaxation [41]. Next, we addressed the question of whether the U-II is involved in penile erection in vivo by using an experimental animal model. Indeed, the measure of cavernous pressure in vivo in rats or mice have been widely used to define the relevance of other pathways in penile erection [36,42]. Intracavernous administration of U-II in anesthetized rats induced a dosedependent increase in ICP implying that the peptide is involved in penile erection. In order to validate the assay, parallel experiments with acetylcholine were performed. Intracavernous administration of acetylcholine caused, as expected, an increase in ICP. Interestingly, whereas intracavernous administration of acetylcholine, as expected, reduced the blood pressure, U-II did not cause any significant change in systemic pressure at all the doses tested. Thus, U-II can be added to the other vasoactive petide such as vasoactive intestinal polypeptide (VIP) or calcitonin generelated peptide in the physiology of erectile function. In fact, the balance between contractant (e.g., noradrenaline, endothelins, angiotensins) and relaxant (e.g., NO, VIP, and related peptides, prostanoids) factors controls the degree of contraction of corpora cavernosa smooth muscle. In particular, VIP has an inhibitory and relaxation-producing effect on strips of HCC tissue and cavernosal vessel in vitro. The role of VIP in vivo has not
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U-II Involvement in Erectile Function been clearly established however its receptors could represent an interesting therapeutic target [43]. Conclusion
HCC possesses U-II receptor that is located predominantly on the endothelium. The receptor is functional and mediates an endotheliumdependent relaxation that involves the L-arginine/NO pathway. Overall these data taken together with the finding that U-II causes an increase in ICP in rats, clearly suggest a role for U-II in the complex regulation of penile erection. However, the understanding of the precise mechanism through that the signal transduction mechanism(s) are initiated by the activation of UT receptor still requires further studies. These observations may help to unravel the complex mechanisms underlying the pathophysiology of human penile erection and may lead to the development of novel therapeutic approaches in the treatment of ED and sexual arousal disorders. Corresponding Author: Giuseppe Cirino, PhD, FBPharmacolS, Via Domenico Montesano, 49, 80131 Napoli, Italy. Tel: +39-081678442; Fax: +39081678403; E-mail:
[email protected] Conflict of Interest: None.
Statement of Authorship
Category 1 (a) Conception and Design Raffaella Sorrentino; Paolo Grieco; Roberta d’Emmanuele di Villa Bianca; Giuseppe Cirino (b) Acquisition of Data Roberta d’Emmanuele di Villa Bianca; Emma Mitidieri; Ciro Coletta; Gianluca Grassia; Fiorentina Roviezzo; Paolo Grieco (c) Analysis and Interpretation of Data Roberta d’Emmanuele di Villa Bianca; Giuseppe Cirino; Raffaella Sorrentino; Ettore Novellino; Ciro Imbimbo
Category 2 (a) Drafting the Article Roberta d’Emmanuele di Villa Bianca; Raffaella Sorrentino; Giuseppe Cirino; Paolo Grieco; Gianluca Grassia (b) Revising It for Intellectual Content Roberta d’Emmanuele di Villa Bianca; Raffaella Sorrentino; Vincenzo Mirone; Ettore Novellino
Category 3 (a) Final Approval of the Completed Manuscript Roberta d’Emmanuele di Villa Bianca; Raffaella Sorrentino; Giuseppe Cirino; Ciro Coletta; Emma Mitidieri; Gianluca Grassia; Vincenzo Mirone; Ciro Imbimbo; Paolo Grieco; Ettore Novellino; Fiorentina Roviezzo References 1 Bern HA, Pearson D, Larson BA, Nishioka RS. Neurohormones from fish tails: The caudal neurosecretory system. I. “Urophysiology” and the caudal neurosecretory system of fishes. Recent Prog Horm Res 1985;41:533–52. 2 Conlon JM, Yano K, Waugh D, Hazon N. Distribution and molecular forms of urotensin II and its role in cardiovascular regulation in vertebrates. J Exp Zool 1996;275:226–38. 3 Ames RS, Sarau HM, Chambers JK, Willette RN, Aiyar NV, Romanic AM, Louden CS, Foley JJ, Sauermelch CF, Coatney RW, Ao Z, Disa J, Holmes SD, Stadel JM, Martin JD, Liu WS, Glover GI, Wilson S, McNulty DE, Ellis CE, Elshourbagy NA, Shabon U, Trill JJ, Hay DW, Ohlstein EH, Bergsma DJ, Douglas SA. Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 1999; 401:282–6. 4 Coulouarn Y, Lihrmann I, Jegou S, Anouar Y, Tostivint HC, Beauvillain J, Conlon JM, Bern HA, Vaudry H. Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord. Proc Natl Acad Sci USA 1998;95:15803–8. 5 Cheung BM, Leung R, Man YB, Wong LY. Plasma concentration of urotensin II is raised in hypertension. J Hypertens 2004;22:1341–4. 6 Douglas SA, Dhanak D, Johns DG. From “gills to pills”: Urotensin-II as a regulator of mammalian cardiorenal function. Trends Pharmacol Sci 2004;25:76–85. 7 Douglas SA, Ashton DJ, Sauermelch CF, Coatney RW, Ohlstein DH, Ruffolo MR, Ohlstein EH, Aiyar NV, Willette RN. Human urotensin-II is a potent vasoactive peptide: Pharmacological characterization in the rat, mouse, dog and primate. J Cardiovasc Pharmacol 2000;36(5 suppl 1):S163–6. 8 Douglas SA, Ohlstein EH. Human urotensin-II, the most potent mammalian vasoconstrictor identified to date, as a therapeutic target for the management of cardiovascular disease. Trends Cardiovasc Med 2000;10:229–37. 9 Camarda V, Rizzi A, Calò G, Gendron G, Perron SI, Kostenis E, Zamboni P, Mascoli F, Regoli D. Effects of human urotensin II in isolated vessels of various species; comparison with other vasoactive agents. Naunyn Schmiedebergs Arch Pharmacol 2002;365:141–9. 10 Douglas SA, Sulpizio AC, Piercy V, Sarau HM, Ames RS, Aiyar NV, Ohlstein EH, Willette RN. Differential vasoconstrictor activity of human urotensin-II in vascular tissue isolated from the rat, mouse, dog, pig, marmoset and cynomolgus monkey. Br J Pharmacol 2000;131:1262–74. 11 MacLean MR, Alexander D, Stirrat A, Gallagher M, Douglas SA, Ohlstein EH, Morecroft I, Polland K. Contractile responses to human urotensin-II in rat and human pulmonary arteries: Effect of endothelial factors and chronic hypoxia in the rat. Br J Pharmacol 2000;130:201–4. 12 Rossowski WJ, Cheng BL, Taylor JE, Datta R, Coy DH. Human urotensin II-induced aorta ring contractions are mediated by protein kinase. C, tyrosine kinases and Rho-kinase: Inhibition by somatostatin receptor antagonists. Eur J Pharmacol 2002;438:159–70.
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