Relaxation Of Human Corpus Cavernosum Research Articles

Erectile dysfunction is associated with defective L-cysteine/hydrogen sulfide pathway in human corpus cavernosum and penile arteries

H2S signaling was proposed to participate in erectile physiology. L-cysteine (CYS)/H2S pathway stimulation causes cGMP-dependent relaxation of human corpus cavernosum (HCC) and penile arteries (HPRA). The aim was to evaluate the impact of ED on CYS/H2S pathway at functional and molecular level in human penile vascular tissues. NaHS- and CYS-induced responses were evaluated in HCC and HPRA from organ donors without ED (NoED, n = 29) and from ED patients undergoing penile prosthesis insertion (n = 45). cGMP accumulation and cystathionine β-synthase and cystathionine γ-lyase expression were also determined.NaHS-induced relaxations were slightly but significantly impaired in HCC but not in HPRA from ED patients. In contrast, CYS-induced relaxations were markedly impaired in HCC (Emax 67.6 ± 4.9% vs 46.2 ± 4.6%, P < 0.01) and HPRA (Emax 80.8 ± 4.0% vs 48.1 ± 8.6%, P < 0.05) from men with ED. Impairment of CYS-induced responses was observed even after separating diabetic ED patients. In HPRA from ED patients, CYS- but not NaHS-induced vasodilation was significantly associated to endothelial function measured as vasodilatory capacity of acetylcholine (ACh) in these preparations (r2 = 0.481, P < 0.01). Impairment of CYS-induced relaxations was related to significant reduction in CYS-induced accumulation of cGMP in cavernosal tissue. Furthermore, the expression of H2S synthesizing enzymes was significantly reduced in HCC from ED patients with respect to NoED. This was confirmed by immunofluorescence in HCC and HPRA sections. ED involves impairment of CYS/H2S pathway in penile vascular tissues associated with decreased expression of H2S generating enzymes, CBS and CSE. These evidences support a therapeutic potential for modulation of CYS/H2S signaling in the management of ED.

MP84-09 DEFECTIVE L-CYSTEINE/HYDROGEN SULFIDE PATHWAY IN HUMAN CORPUS CAVERNOSUM AND PENILE ARTERIES FROM MEN WITH ERECTILE DYSFUNCTION

You have accessJournal of UrologySexual Function/Dysfunction: Basic Research & Pathophysiology (MP84)1 Apr 2020MP84-09 DEFECTIVE L-CYSTEINE/HYDROGEN SULFIDE PATHWAY IN HUMAN CORPUS CAVERNOSUM AND PENILE ARTERIES FROM MEN WITH ERECTILE DYSFUNCTION Alejandro Sevilleja-Ortiz, José M. La Fuente, Juan Ignacio Martínez-Salamanca*, Esaú Fernández-Pascual, Joaquín Carballido, and Javier Angulo Alejandro Sevilleja-OrtizAlejandro Sevilleja-Ortiz More articles by this author , José M. La FuenteJosé M. La Fuente More articles by this author , Juan Ignacio Martínez-Salamanca*Juan Ignacio Martínez-Salamanca* More articles by this author , Esaú Fernández-PascualEsaú Fernández-Pascual More articles by this author , Joaquín CarballidoJoaquín Carballido More articles by this author , and Javier AnguloJavier Angulo More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000976.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Hydrogen sulfide (H2S) signaling has been proposed to participate in erectile physiology. L-cysteine (CYS)/H2S pathway stimulation causes cGMP-dependent relaxation of human corpus cavernosum (HCC) and penile arteries (HPRA). The aim of this work was to evaluate the impact of erectile dysfunction (ED) on CYS/H2S pathway at functional and molecular level in human penile vascular tissues. METHODS: NaHS- and CYS-induced responses were evaluated in HCC and HPRA from organ donors without notice of ED (NoED, n=29) and from ED patients undergoing penile prosthesis insertion (n=45). cGMP accumulation and cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) expressions were also determined in these tissues. RESULTS: NaHS-induced relaxations were slightly but significantly impaired in HCC (pEC50 4.58±0.09 vs. 4.19±0.07, p<0.01) but not in HPRA (pEC50 4.16±0.10 vs. 4.00±0.07) from ED patients. In contrast, CYS-induced relaxations were markedly impaired in both HCC (Emax 67.6±4.9% vs 46.2±4.6%, p<0.01) and HPRA (Emax 80.8±4.0% vs 48.1±8.6%, p<0.05) from men with ED. Impairment of CYS-induced responses was observed even after separating diabetic ED patients. In HPRA from ED patients, CYS- but not NaHS-induced vasodilation was significantly associated to endothelial function measured as vasodilatory capacity of acetylcholine (ACh) in these preparations (pEC25 CYS vs. Emax ACh: r2=0.481, p<0.01). Impairment of CYS-induced relaxations was related to significant reduction in CYS-induced accumulation of cGMP in cavernosal tissue. Furthermore, the expression of H2S synthesizing enzymes CBS and CSE, as determined by Western blot, was significantly reduced in HCC from ED patients with respect to NoED tissues. This diminished expression of CBS and CSE was clearly manifested in immunofluorescence assays in HCC and HPRA sections. CONCLUSIONS: ED involves impairment of CYS/H2S pathway in penile vascular tissues associated with decreased expression of H2S generating enzymes, CBS and CSE. These evidences support a therapeutic potential for modulation of CYS/H2S signaling in the management of ED. Source of Funding: None © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e1268-e1268 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alejandro Sevilleja-Ortiz More articles by this author José M. La Fuente More articles by this author Juan Ignacio Martínez-Salamanca* More articles by this author Esaú Fernández-Pascual More articles by this author Joaquín Carballido More articles by this author Javier Angulo More articles by this author Expand All Advertisement PDF downloadLoading ...

L-cysteine/hydrogen sulfide pathway induces cGMP-dependent relaxation of corpus cavernosum and penile arteries from patients with erectile dysfunction and improves arterial vasodilation induced by PDE5 inhibition

The aim was to evaluate and characterize H2S-induced relaxation of human corpus cavernosum (HCC) and penile resistance arteries (HPRA) from patients with erectile dysfunction (ED). HCC and HPRA were obtained from men with ED at the time of penile prosthesis insertion. H2S-mediated relaxations were evaluated by exposing these tissues to the stable analogue, NaHS, and to the precursor of H2S, L-cysteine (CYS). The effects of NaHS and CYS were also evaluated on cGMP accumulation in HCC and on acetylcholine- and sildenafil-mediated relaxations in HCC and HPRA. NaHS consistently relaxed HPRA and HCC and more potently than human prostate and bladder. NaHS-induced relaxations in HCC and HPRA were unaffected by the ATP-sensitive K+-channel blocker, glibenclamide or the NO synthase inhibitor, L-NAME, slightly reduced by the Ca2+-activated K+-channel blocker, tetraethylammonium, and markedly inhibited by the soluble guanylyl cyclase inhibitor, ODQ. NaHS caused a cGMP increase in HCC that was inhibited by ODQ. CYS produced relaxations of HCC and HPRA that were sensitive to ODQ and to inhibition of the H2S synthesizing enzymes, cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS). CYS also increased cGMP in HCC. In contrast to NaHS, CYS-induced relaxations were prevented by endothelium removal in HPRA. Only in HPRA, treatment with CYS (30 μM) potentiated acetylcholine- and sildenafil-induced relaxations. This effect was prevented by CSE/CBS inhibition and by removing the endothelium. Exogenous and endogenous H2S relaxes HCC and HPRA from ED patients through cGMP accumulation and potentiates vasodilatory capacity of PDE5 inhibition, supporting the therapeutic potential of modulating H2S pathway.

931 - Testosterone associated relaxation of human corpus cavernosum of patients with erectile dysfunction: Are non genomic pathways involved?

931 - Testosterone associated relaxation of human corpus cavernosum of patients with erectile dysfunction: Are non genomic pathways involved?

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

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.

Additive effects of the Rho kinase inhibitor Y-27632 and vardenafil on relaxation of the corpus cavernosum tissue of patients with erectile dysfunction and clinical phosphodiesterase type 5 inhibitor failure.

To evaluate the expression of the Rho/Rho-associated protein kinase (ROCK) pathway in the corpus cavernosum of patients with severe erectile dysfunction (ED) compared with healthy human corpus cavernosum, and to test the functional effects of two Rho kinase inhibitors (RKIs) on erectile tissue of patients with severe ED, which did not respond to phosphodiesterase type 5 inhibitors (PDE5Is). Human corpus cavernosum samples were obtained after consent from men undergoing penile prosthesis implantation (n = 7 for organ bath experiments, n = 17 for quantitative PCR [qPCR]). Potent control subjects (n = 5) underwent penile needle biopsy. qPCR was performed for the expression of RhoA and ROCK subtypes 1 and 2. Immunohistochemistry staining against ROCK and α smooth muscle actin (αSMA) was performed on the corpus cavernosum of patients with ED. Tissue strips were precontracted with phenylephrine and incubated with 1 μm of the PDE5I vardenafil or with DMSO (control). Subsequently, increasing concentrations of the RKIs azaindole or Y-27632 were added, and relaxation of tissue was quantified. The expression of ROCK1 was unchanged (P > 0.05), while ROCK2 (P < 0.05) was significantly upregulated in patients with ED compared with controls. ROCK1 and ROCK2 protein colocalized with αSMA, confirming the presence of this kinase in cavernous smooth muscle cells and/or myofibroblasts. After incubation with DMSO, 10 μm azaindole and 10 μm Y-27632 relaxed precontracted tissues with 49.5 ± 7.42% (P = 0.1470 when compared with vehicle) and 85.9 ± 10.3% (P = 0.0016 when compared with vehicle), respectively. Additive effects on relaxation of human corpus cavernosum were seen after preincubation with 1 μm vardenafil. The RKI Y-27632 causes a significant relaxation of corpus cavernosum in tissue strips of patients with severe ED. The additive effect of vardenafil and Y-27632 shows that a combined inhibition of Rho-kinase and phosphodiesterase type 5 could be a promising orally administered treatment for severe ED.

MP52-05 BILATERAL CAVERNOUS NERVE INJURY INDUCES TEMPORAL INCREASE OF TNF-α AND RECRUITMENT OF MACROPHAGES IN THE MAJOR PELVIC GANGLION IN VIVO.

INTRODUCTION AND OBJECTIVES: In addition to a loss of nitrergic function, cavernous nerve injury (CNI) in rats and radical prostatectomy in men result in an enhancement of adrenergic-neurogenic contractions of cavernosal tissue. Our aim was to evaluate the modulation of a-adrenergic system as a strategy to relieve erectile dysfunction (ED) and functional cavernosal alterations induced by CNI in rats. The effects of a-adrenergic blockade on neurogenic relaxation of human corpus cavernosum (HCC) were also evaluated. METHODS: Bilateral crush CNI (BCNI) was induced in anesthetized rats that were randomly selected to receive or not the non-selective a-blocker, phentolamine (1 mg/kg/day; PHENT) or the selective a1A-blocker, silodosine (0.1 mg/kg/day; SILOD) in drinking water for 4 weeks. Erectile responses to cavernosal nerve stimulation and neurogenic responses of corpus cavernosum to electrical field stimulation (EFS) in organ chambers were then evaluated. HCC from ED patients were obtained at the time of penile prosthesis implantation and nitrergic responses to EFS were determined. RESULTS: Chronic treatment with SILOD significantly improved erectile responses, partially recovered nitrergic relaxations and normalized neurogenic contractions (Table). Chronic a-blockade with PHENT or SILOD allowed for a significant potentiation of erectile responses in BCNI rats after acute administration of the PDE5 inhibitor tadalafil (0.3 mg/kg; i.v.). Treatment of HCC with PHENT (1 mM) slightly increased nitrergic relaxations and enhanced the potentiating effects of tadalafil (30 nM) on these responses. CONCLUSIONS: a-adrenergic modulation, especially selective a1A-blockade, improves erectile and cavernosal functions after BCNI. Modulation of adrenergic system, mainly in combination strategies, could have a role in the management of ED after RP.

MP52-04 SELECTIVE α1A-ADRENERGIC MODULATION ENHANCES ERECTILE RESPONSES IN RATS AFTER CAVERNOUS NERVE INJURY AND IMPROVES NEUROGENIC AND PDE5 INHIBITOR RESPONSES IN HUMAN AND RAT CAVERNOSAL TISSUE

INTRODUCTION AND OBJECTIVES: In addition to a loss of nitrergic function, cavernous nerve injury (CNI) in rats and radical prostatectomy in men result in an enhancement of adrenergic-neurogenic contractions of cavernosal tissue. Our aim was to evaluate the modulation of a-adrenergic system as a strategy to relieve erectile dysfunction (ED) and functional cavernosal alterations induced by CNI in rats. The effects of a-adrenergic blockade on neurogenic relaxation of human corpus cavernosum (HCC) were also evaluated. METHODS: Bilateral crush CNI (BCNI) was induced in anesthetized rats that were randomly selected to receive or not the non-selective a-blocker, phentolamine (1 mg/kg/day; PHENT) or the selective a1A-blocker, silodosine (0.1 mg/kg/day; SILOD) in drinking water for 4 weeks. Erectile responses to cavernosal nerve stimulation and neurogenic responses of corpus cavernosum to electrical field stimulation (EFS) in organ chambers were then evaluated. HCC from ED patients were obtained at the time of penile prosthesis implantation and nitrergic responses to EFS were determined. RESULTS: Chronic treatment with SILOD significantly improved erectile responses, partially recovered nitrergic relaxations and normalized neurogenic contractions (Table). Chronic a-blockade with PHENT or SILOD allowed for a significant potentiation of erectile responses in BCNI rats after acute administration of the PDE5 inhibitor tadalafil (0.3 mg/kg; i.v.). Treatment of HCC with PHENT (1 mM) slightly increased nitrergic relaxations and enhanced the potentiating effects of tadalafil (30 nM) on these responses. CONCLUSIONS: a-adrenergic modulation, especially selective a1A-blockade, improves erectile and cavernosal functions after BCNI. Modulation of adrenergic system, mainly in combination strategies, could have a role in the management of ED after RP.

MP47-10 NO/CGMP-MEDIATED RELAXATION INDUCED BY INSULIN IN HUMAN CORPUS CAVERNOSUM AND PENILE ARTERIES IS IMPAIRED IN ERECTILE DYSFUNCTION.

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2014MP47-10 NO/CGMP-MEDIATED RELAXATION INDUCED BY INSULIN IN HUMAN CORPUS CAVERNOSUM AND PENILE ARTERIES IS IMPAIRED IN ERECTILE DYSFUNCTION. Juan I. Martínez-Salamanca, José M. La Fuente, Argentina Fernández, Eduardo Martínez-Salamanca, Pepe Cardoso, Joaquín Carballido, and Javier Angulo Juan I. Martínez-SalamancaJuan I. Martínez-Salamanca More articles by this author , José M. La FuenteJosé M. La Fuente More articles by this author , Argentina FernándezArgentina Fernández More articles by this author , Eduardo Martínez-SalamancaEduardo Martínez-Salamanca More articles by this author , Pepe CardosoPepe Cardoso More articles by this author , Joaquín CarballidoJoaquín Carballido More articles by this author , and Javier AnguloJavier Angulo More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1463AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives In addition to regulate glucose homeostasis, insulin has been demonstrated to influence vascular function at local level. However, the influence of insulin in human erectile tissue is not well defined. The aim of this work was to characterize the effects of insulin in human corpus cavernosum (HCC) and penile resistance arteries (HPRA) analyzing its interactions with NO/cGMP pathway and the influence of erectile dysfunction (ED). Methods HCC and HPRA were obtained from organ donors (NoED) and patients with erectile dysfunction (ED) who gave informed consent at the time of penile prosthesis implantation. HCC and HPRA were mounted in organ chambers and wire myographs, respectively, for isometric tension recording. cGMP was determined in HCC homogenates by ELISA. Results Insulin caused concentration-dependent relaxation of HCC (n=20) and vasodilation of HPRA (n=32) (pD2 7.40±0.33 and 7.21±0.32, respectively; Emax 64.0±6.1% and 59.3±5.3%, respectively). Insulin-induced relaxation of HCC was impaired in ED patients (53.0±7.4%) with respect to NoED (80.7±7.5%, p<0.01). Responses in NoED were inhibited by guanylyl cyclase inhibition with 20μM ODQ while responses in ED were potentiated by PDE5 inhibition with 30nM tadalafil. NO/cGMP contribution was confirmed by cGMP elevation (3.18±0.54 fold increase) after exposure to 1μM insulin. Involvement of endothelium was suggested by the marked difference between vasodilation in HPRA with <25% relaxation to 10μM acetylcholine (ACh) and HPRA with >40% relaxation to ACh (47.0±6.8 vs. 68.7±7.5, p<0.01). Treatment with insulin (10nM) improved endothelium-dependent relaxation in HCC, especially in ED patients with diabetes (52.3±11.5% vs. 81.1±5.4%, p<0.05). Conclusions Insulin causes human penile smooth muscle relaxations that are mediated by NO/cGMP pathway, contributed by endothelium, and impaired by the presence of ED. Insulin also participates in the regulation of endothelial function in erectile tissue. Altered regulation of penile smooth muscle tone by insulin could contribute to ED in metabolic disorders. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e522 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Juan I. Martínez-Salamanca More articles by this author José M. La Fuente More articles by this author Argentina Fernández More articles by this author Eduardo Martínez-Salamanca More articles by this author Pepe Cardoso More articles by this author Joaquín Carballido More articles by this author Javier Angulo More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Activation of P2Y1 and P2Y2 nucleotide receptors by adenosine 5′-triphosphate analogues augmented nerve-mediated relaxation of human corpus cavernosum

INTRODUCTION: Adenosine 5'-triphosphate (ATP) is a ubiquitous cellular energy source. We evaluated the effect of ATP and its analogues on nonadrenergic and noncholinergic relaxation in precontracted human corpus cavernosal smooth muscle (HCCSM). METHODS: We obtained specimens of human corpus cavernosum (HCC) from patients undergoing penile prosthesis surgery (patient age 46-70 yr, n = 17) with prior approval from the local institutional review board. Isolated HCC strips were placed in organ baths containing Krebs solution and functional experiments were conducted. Immunohistochemical localization studies were performed to establish the presence of purinergic P2X1, P2Y1 and P2Y2 receptors in HCC. RESULTS: The amplitude of relaxation induced by electrical-field stimulation (EFS) on HCC was significantly increased after exposure to ATP (P2X and P2Y agonists), 2-MeSATP (P2Y1 agonist), and uridine 5' triphosphate (P2Y2 agonist), but not alpha,beta-methylene ATP (P2X1 agonist). The P2X1 antagonist pyridoxal-5'-phosphate-6-azophenyl-2', 4'-disulfonate, and the nonspecific P2Y antagonist, reactive blue 2, did not inhibit the potentiated response of EFS on HCC. Although immunoreactivity for both P2Y1 and P2Y2 receptors was localized abundantly in HCC, there was only low-level immunostaining for the P2X1 receptor. CONCLUSION: These data demonstrate that nerve-mediated relaxation of HCCSM strips precontracted with phenylephrine in organ bath preparations is amplified by stimulating purinergic P2Y1 and P2Y2 receptors. Although nucleotides are important regulators of HCCSM tone, these observations suggest an independent purinergic relaxing mechanism in the HCC, separate from the better known nitrergic system.

Erectile Dysfunction

Erectile Dysfunction

Endogenous Urotensin II Selectively Modulates Erectile Function through eNOS

BackgroundUrotensin II (U-II) is a cyclic peptide originally isolated from the neurosecretory system of the teleost fish and subsequently found in other species, including man. U-II was identified as the natural ligand of a G-protein coupled receptor, namely UT receptor. U-II and UT receptor are expressed in a variety of peripheral organs and especially in cardiovascular tissue. Recent evidence indicates the involvement of U-II/UT pathway in penile function in human, but the molecular mechanism is still unclear. On these bases the aim of this study is to investigate the mechanism(s) of U-II-induced relaxation in human corpus cavernosum and its relationship with L-arginine/Nitric oxide (NO) pathway.Methodology/Principal FindingsHuman corpus cavernosum tissue was obtained following in male-to-female transsexuals undergoing surgical procedure for sex reassignment. Quantitative RT-PCR clearly demonstrated the U-II expression in human corpus cavernosum. U-II (0.1 nM–10 µM) challenge in human corpus cavernosum induced a significant increase in NO production as revealed by fluorometric analysis. NO generation was coupled to a marked increase in the ratio eNOS phosphorilated/eNOS as determined by western blot analysis. A functional study in human corpus cavernosum strips was performed to asses eNOS involvement in U-II-induced relaxation by using a pharmacological modulation. Pre-treatment with both wortmannin or geldanamycinin (inhibitors of eNOS phosphorylation and heath shock protein 90 recruitment, respectively) significantly reduced U-II-induced relaxation (0.1 nM–10 µM) in human corpus cavernosum strips. Finally, a co-immunoprecipitation study demonstrated that UT receptor and eNOS co-immunoprecipitate following U-II challenge of human corpus cavernosum tissue.Conclusion/SignificanceU-II is endogenously synthesized and locally released in human corpus cavernosum. U-II elicited penile erection through eNOS activation. Thus, U-II/UT pathway may represent a novel therapeutical target in erectile dysfunction.

Effect of vardenafil on nitric oxide synthase expression in the paraventricular nucleus of rats without sexual stimulation

Vardenafil hydrochloride (HCl) is a potent and selective phosphodiesterase type-5 (PDE-5) inhibitor that enhances nitric oxide (NO)-mediated relaxation of human corpus cavernosum and NO-induced rabbit penile erection, and enhances erectile function in patients. In the present study, the effect of vardenafil on nitric oxide synthase (NOS) and neuronal NOS expressions in the paraventricular nucleus (PVN) of rats without sexual stimulation was investigated using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal NOS (nNOS) immunohistochemistry and western blot analysis. The present results showed that NOS and nNOS expression in the PVN was increased by vardenafil treatment as the dose- and duration-dependently without sexual stimulation. The phosphodiesterase type-5 inhibitor, vardenafil, augmented NOS expression in the brain without sexual stimulation. The present study suggests that sexual behaviour can be directly modulated by neurotransmitters such as nitric oxide.

Testosterone Might Cause Relaxation of Human Corpus Cavernosum by Potassium Channel Opening Action

To investigate the effect of testosterone on contractile tone of endothelium-denuded human corpus cavernosum strips. Human studies designed to examine a possible relaxant effect of testosterone on corpus cavernosal circulation are lacking. Testosterone (0.1-300 microM) was added cumulatively to organ baths after precontraction of isolated human corpus cavernosum strips (n = 5) with KCl (45 mM). Testosterone-induced responses were tested in the presence of nonselective, large, conductance Ca(2+)-activated and voltage-sensitive K(+) channel inhibitor tetraethylammonium (1 mM), adenosine triphosphate-sensitive K(+) channel inhibitor glibenclamide (10 microM), voltage-dependent inward rectifier K(+) channel inhibitor barium chloride (30 microM) and voltage-sensitive K(+) channel inhibitor 4-aminopyridine (1 mM). Testosterone (0.1-300 microM) produced relaxation in human corpus cavernosum (maximum relaxation 65.4% +/- 3.3% of KCl-induced contraction) that reached a maximum at a concentration of 300 microM. Testosterone-induced relaxation was significantly attenuated by glibenclamide, but it was not affected by the other K(+) channel inhibitors (tetraethylammonium, barium chloride, or 4-aminopyridine). Testosterone might induce relaxation in human isolated corpora cavernosa strips by activation of smooth muscle adenosine triphosphate-sensitive K(+) channels. This finding suggests that testosterone, in addition to its known endothelial action, might regulate erectile function locally by its action on the smooth muscle of the human corpus cavernosum.

Optimizing Nitric Oxide Production by Time Dependent L-Arginine Administration in Isolated Human Corpus Cavernosum

We investigated the relaxant effects of repetitive administration of L-arginine, the substrate for nitric oxide, at hourly intervals and elucidated its mechanism of action in human corpus cavernosum. Samples of human corpus cavernosum were suspended in an organ chamber for measurements of isometric tension. After precontraction with phenylephrine (10 microM), concentration-response curves were performed for L-arginine at 2-hour intervals (1 to 10 hours). Underlying mechanisms of relaxation were evaluated by inhibitory and stimulatory agents. After a brief incubation period of 1 to 4 hours L-arginine (0.1 to 1,000 microM) but not D-arginine induced minor changes in HCC. In contrast, when incubation time was increased to 6 to 10 hours L-arginine evoked detectable human corpus cavernosum relaxation proportional to concentration and time. Relaxation was significantly attenuated by the nitric oxide synthase inhibitor L-NAME, the blocker of soluble guanylyl cyclase ODQ and the blocker of small conductance Ca2+ activated K+ channels apamin, and partially by the inducible nitric oxide synthase inhibitor aminoguanidine and the cyclic guanosine 5'-monophosphate dependent protein kinase G inhibitor Rp-8-pCPT-cGMPS. Relaxation was potentiated in the presence of the membrane permeable cyclic guanosine 5'-monophosphate analogue 8-bromo-cyclic guanosine 5'-monophosphate, the Rho-kinase inhibitor Y-27632 and the phosphodiesterase-5 inhibitor sildenafil. These observations demonstrate that L-arginine induces slow and prolonged relaxation of human corpus cavernosum. This may occur by restoring the endogenous amino acid pool for nitric oxide synthesis and by nitric oxide-soluble guanylyl cyclase-protein kinase G signaling involving the activation of KCa channels or by inhibiting the up-regulated RhoA/Rho-kinase pathway. The use of sildenafil combined with L-arginine further facilitates erections and it may benefit men with more severe erectile dysfunction.

Enhanced Thromboxane Receptor-Mediated Responses and Impaired Endothelium-Dependent Relaxation in Human Corpus Cavernosum from Diabetic Impotent Men: Role of Protein Kinase C Activity

We have evaluated the influence of protein kinase C (PKC) activity on penile smooth muscle tone in tissues from diabetic and nondiabetic men with erectile dysfunction. Human corpus cavernosum (HCC) strips were obtained from impotent diabetic and nondiabetic men at the time of penile prosthesis implantation and studied in organ chambers. Contractility responses to a prostanoid precursor, to prostanoids, and to the endothelium-dependent vasodilator acetylcholine were studied. Arachidonic acid (AA; 100 microM) caused cyclooxygenase-dependent relaxation of HCC. This relaxation was impaired in diabetic tissues and normalized by blocking thromboxane (TP) receptors with 20 nM [1S-[1alpha,2alpha(Z),3alpha,4alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ29548). Diabetes did not affect prostaglandin (PG)E(1)-induced relaxation, but it reduced relaxation induced by the PGE(1) metabolite PGE(0). This effect was related to an interaction of PGE(0) with TP receptors. Diabetic tissues had reduced endothelium-dependent relaxation, which was partially improved by SQ29548 and completely normalized by the PKC inhibitor 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X; 1 microM). In HCC from nondiabetic patients, treatment with the PKC activator phorbol-12,13-dibutyrate (0.3 microM) significantly attenuated endothelium-dependent relaxation, an effect prevented by coadministration of GF109203X. Tissues from diabetic patients had enhanced sensitivity to the contractile effects of the TP receptor agonist 9,11-dideoxy-9alpha,11alpha-epoxymethano PGF(2alpha) (U46619) (EC(50) = 0.65 +/- 0.42 and 6.01 +/- 2.28 nM in diabetic and nondiabetic patients, respectively). Inhibition of PKC with 1 microM GF109203X, prevented diabetes-induced hypersensitivity to U46619-induced contractions (EC(50) = 8.55 +/- 3.12 microM). Overactivity of PKC in diabetes is responsible for enhanced contraction and reduced endothelium-dependent relaxation of HCC smooth muscle. Such alterations can result in erectile dysfunction.

The neuronal and endothelium-dependent relaxing responses of human corpus cavernosum under physiological oxygen tension last longer than previously expected.

Intracavernosal oxygen tension varies greatly in the process of erection. Blood extracted from the human penis demonstrates an increase from approximately 30 mmHg Po(2) in the flaccid state to 100 mmHg in the erect state of the penis. In the present study, using these levels as a guide, we investigate how the NO-dependent relaxation of human corpus cavernosum changed under physiological oxygen tensions ranging from approximately 30 to 100 mmHg. Human penile tissue specimens were obtained at penile surgery with informed consent from the patients. The preparations were mounted in Krebs solution in an organ bath and the isometric tension was recorded. Krebs solutions of various oxygen tensions were prepared by bubbling 5% CO(2) in N(2) and O(2). The NO-dependent relaxation caused by electrical field stimulation (EFS) and acetylcholine (ACh) was studied, and the amplitude and duration of relaxation evaluated. The amplitude of relaxation induced by EFS was significantly decreased under physiological oxygen tension conditions (P < 0.01). The duration of the relaxant response induced by EFS and ACh was significantly prolonged in physiological oxygen tension conditions than in high oxygen tension (P < 0.01). However, there was no correlation between the duration of relaxation induced by EFS and each physiological oxygen tension level. The duration of relaxation induced by ACh was most prolonged at 60-69 mmHg oxygen tension. Physiologically, the effect of NO may last longer than was previously thought. In addition, it would seem that there is an optimal physiological oxygen tension for maximum ACh-induced relaxation.

EFFECTS OF SILDENAFIL ON THE RELAXATION OF HUMAN CORPUS CAVERNOSUM TISSUE IN VITRO AND ON THE ACTIVITIES OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ISOZYMES

Purpose Sildenafil, an inhibitor of cGMP-specific phosphodiesterase 5 (PDE5), is currently undergoing evaluation as an oral therapy for penile erectile dysfunction. The aims of this study were to investigate the mechanism of action of sildenafil on the neurogenic relaxation of human corpus cavernosum (HCC) in vitro and to determine the activity of sildenafil against a full range of PDE isozymes. Materials and Methods Strips of HCC tissue were precontracted with phenylephrine. Relaxation responses resulting from electrical field stimulation (EFS) were then determined in the presence and absence of sildenafil. The effects of sildenafil on PDE1 to 5 prepared from human tissues and PDE6 from bovine retina were determined by measuring the conversion of [( 3) H]-cGMP or [( 3) H]-cAMP to their respective [( 3) H]-5′-mononucleotides. Results Sildenafil (0.001 to 1 micro M) enhanced the EFS-induced, nitric oxide (NO) dependent, relaxation of HCC in a concentration-dependent manner to a maximum of 3 times the pretreatment level at 1 micro M sildenafil. Compared with zaprinast, an early PDE5 inhibitor, sildenafil was approximately 240-fold more potent, inhibiting PDE5 from HCC with a geometric mean IC 50 of 3.5 nM. For sildenafil, IC 50 values for inhibition of PDE1 to 4 were 80 to more than 8500 times greater than that for PDE5 and the IC 50 for PDE6 (33 nM) was approximately 9-fold greater. Conclusions The data support the proposal that enhancement of penile erection by sildenafil in patients with erectile dysfunction involves potentiation of the NO-stimulated cGMP signal mediating relaxation of cavernosal smooth muscle during sexual stimulation. Sildenafil is a potent inhibitor of PDE5 from HCC, with high selectivity for PDE5 relative to other PDE isozymes.

Pharmacological characterization of kinin-induced relaxation of human corpus cavernosum.

To characterize the kinin receptor subtype involved in the relaxation of human isolated corpus cavernosum (HCC) induced by bradykinin (BK), Lys-bradykinin (Lys-BK), Met-Lys-bradykinin (Met-Lys-BK) and des-Arg9-bradykinin, and to investigate whether the kinin-induced relaxation of HCC results from the stimulation of nonadrenergic, noncholinergic (NANC) neurons supplying the cavernosal tissue. Excised HCC tissues were immediately placed in Krebs solution and kept at 4 degrees C until use (never > 24 h after removal). HCC was cut in strips of approximately 2 cm, suspended in a cascade system and superfused with oxygenated and warmed Krebs solution at 5 mL/min. After equilibration for approximately 90 min, noradrenaline (3 micromol/L) was infused to induce a submaximal contraction of the HCC strips. The release of cyclo-oxygenase products was prevented by infusing indomethacin (6 micromol/L). HCC strips were calibrated by injecting a single bolus of the nitrovasodilator glyceryl trinitrate (GTN) and the sensitivity of the tissues adjusted electronically to be similar. The agonists (kinins, histamine and acetylcholine) were injected as a single bolus (up to 100 microL) and the relaxation of HCC expressed as a percentage of the submaximal relaxation induced by GTN. Bradykinin, Lys-BK and Met-Lys-BK significantly relaxed the HCC tissues; on a molar basis, there was no statistical difference among the degrees of relaxation induced by these peptides. The B1 kinin receptor agonist des-Arg9-bradykinin had no effect on the HCC. The infusion of the B2 kinin receptor antagonist Hoe 140 (50 nmol/L) virtually abolished the relaxation induced by BK, Lys-BK and Met-Lys-BK without affecting those induced by acetylcholine and histamine. The infusion of the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester increased the tone of the HCC tissues and significantly reduced (P < 0.01) the relaxation induced by BK (74%), Lys-BK (90%), Met-Lys-BK (87%) and acetylcholine (89%) without affecting those induced by GTN. The subsequent infusion of L-arginine (300 micromol/L) partially reversed the increased tone and significantly (P < 0.01) restored the relaxation induced by BK, Lys-BK and Met-Lys-BK. The results were similar with the novel guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3,-alquinoxalin-1-one] which reduced by > 95% (P < 0.01) the relaxation induced by BK, Lys-BK, Met-Lys-BK, acetylcholine and GTN. The infusion of the sodium-channel blocker tetrodotoxin had no significant effect on the BK-, GTN- and acetylcholine-induced relaxation of HCC. This study clearly showed the existence of functional B2 kinin receptors in human erectile tissues that when activated lead to the release of NO and hence relaxation of the HCC tissues. As tetrodotoxin failed to affect the kinin-induced relaxation of HCC strips, it is likely that these peptides release NO from the endothelium of sinusoidal capillaries rather than from neuronal sources supplying the cavernosal tissue. Although tissue kallikreins and their components have been found in the male reproductive system, the physiopathological importance of these findings has yet to be elucidated.

Nitric oxide and cyclic GMP formation following relaxant nerve stimulation in isolated human corpus cavernosum.

To investigate further the role of the nitric oxide (NO)-cyclic GMP pathway as the mediator of relaxant neurotransmission in human corpus cavernosum and to establish whether impaired activity of this pathway contributes to the pathophysiology of impotence. Samples of cavernosal tissue were obtained from 59 men undergoing penile operations. The controls comprised four men with penile carcinoma and 17 with Peyronie's disease. Of the impotent men, 35 had clinical evidence of penile vascular disease on pre-operative investigation, whilst three had non-vascular impotence. Each biopsy was divided into two strips which were then suspended under tension in organ bath chambers. The relaxant innervation of one strip of each pair was stimulated electrically whilst the other strip was left unstimulated. The formation of NO and cyclic GMP was calculated by comparing their respective tissue content in the stimulated and unstimulated strips. Overall, stimulation of the relaxant innervation produced significant increases in the tissue content of both NO and cyclic GMP. Incubation with an inhibitor of NO biosynthesis abolished the mechanical relaxant response and the formation of both NO and cyclic GMP. The magnitude of relaxant response and the formation of NO was diminished in tissue from men with vascular impotence compared to controls. The increase in cyclic GMP content was similar in both these groups. Relaxant response, NO formation and cyclic GMP formation in tissue from men with non-vascular impotence was similar to controls. This study provides further evidence that the NO-cyclic GMP pathway acts as the mediator of nerve-evoked smooth muscle relaxation in human corpus cavernosum. Diminished NO formation following relaxant nerve stimulation may account for impaired relaxant responses found in tissue from men with vascular impotence and may contribute to the cause of their erectile dysfunction.