Nebivolol Dilates Human Penile Arteries and Reverses Erectile Dysfunction in Diabetic Rats through Enhancement of Nitric Oxide Signaling

IntroductionTraditional beta-blockers have sometimes been associated with erectile dysfunction (ED). Nebivolol is a cardioselective β₁-adrenoceptor antagonist that promotes vasodilation through a nitric oxide (NO)-dependent mechanism.AimWe evaluated the effects of nebivolol on the NO/cyclic guanosine monophosphate (cGMP) signaling pathway, on erectile function and dysfunction, and in human penile vascular tissues.MethodsErectile response to cavernosal nerve electrical stimulation in control and diabetes-induced ED rats were evaluated, along with serum nitrite/nitrate (NOx) concentration and plasma/tissue cGMP levels. Endothelium-dependent and sildenafil-induced relaxation of isolated human corpus cavernosum (HCC) and human penile resistance arteries (HPRA) were also determined.Main Outcome MeasuresThe effects of nebivolol on erectile function and dysfunction and on NO/cGMP-mediated responses.ResultsTreatment with nebivolol significantly potentiated erectile response in control rats, regardless of its effects on blood pressure. Nebivolol increased NOx and plasma cGMP by 3-fold and 2.75-fold, respectively, and significantly augmented the elevation of plasma cGMP produced by sildenafil. Nebivolol enhanced endothelium-dependent and sildenafil-induced relaxations of HCC tissue, and produced endothelium-dependent vasodilation of HPRA. Nebivolol, but not atenolol, significantly improved erectile response in diabetic rats (51.6%, 53.2%, and 87.1% of response at 3 Hz in nondiabetic rats, for vehicle-treated, atenolol-treated, and nebivolol-treated diabetic rats, respectively); after sildenafil administration, ED was completely reversed in nebivolol-treated diabetic rats (69.6% and 112% for diabetic rats treated with sildenafil and nebivolol plus sildenafil, respectively). Accordingly, nebivolol restored systemic NOx levels and cGMP content in penile tissue from these animals.ConclusionsNebivolol in vivo activated the NO/cGMP pathway, enhanced erectile response and reversed ED in diabetic rats. Moreover, nebivolol in vitro potentiated NO/cGMP-mediated relaxation of human erectile tissues. These effects may account for the low incidence of ED in nebivolol-treated hypertensive patients. Nebivolol therefore may have utility in the treatment of ED, particularly ED associated with diabetes. Angulo J, Wright HM, Cuevas P, González-Corrochano R, Fernández A, Cuevas B, La Fuente JM, Gupta S, and de Tejada IS. Nebivolol dilates human penile arteries and reverses erectile dysfunction in diabetic rats through enhancement of nitric oxide signaling.     

Chronic Treatment with an Oral Rho‐Kinase Inhibitor Restores Erectile Function by Suppressing Corporal Apoptosis in Diabetic Rats

IntroductionIt has been suggested that the up‐regulation of the contractile RhoA/Rho‐kinase (ROCK) signaling pathway is one of the important mechanisms for diabetes‐associated erectile dysfunction (ED). However, the exact role of RhoA/ROCK signaling in the pathogenesis of diabetes‐related ED has not been fully delineated.AimTo determine whether the RhoA/ROCK pathway is involved in the regulation of corporal apoptosis and whether administration of insulin or fasudil, a specific ROCK inhibitor, could ameliorate ED in streptozotocin‐induced diabetic rats.Main Outcome MeasuresAt 16 weeks after diabetes induction, erectile function was assessed by cavernous nerve stimulation. Penile tissue was assessed for apoptosis with terminal deoxynucleotidyl transferase‐mediated 2′‐deoxyuridine 5′‐triphosphate (dUTP) nick end labeling assay. Expression of myosin phosphatase target subunit 1 (MYPT1), protein kinase B (Akt), and phospho‐endothelial nitric oxide synthase (eNOS) were evaluated by Western blot. Immunohistochemical study was carried out for smooth muscle alpha‐actin, B‐cell leukemia/lymphoma 2 (Bcl‐2), and Bcl‐2‐associated X Protein (Bax). Activity of caspase‐3 and phosphatase and tensin homolog deleted on chromosome ten (PTEN) was also determined.MethodsMale Sprague‐Dawley rats (8 weeks old) were randomly divided into four groups: age‐matched controls, diabetic controls, and diabetic rats treated with insulin (10 U/day, subcutaneous injection) or fasudil (30 mg/kg/day, oral) for the last 4 weeks of the 16 weeks after diabetes induction.ResultsDiabetic rats showed impairment of erectile function, increased MYPT1 phosphorylation, and corporal apoptosis. Expression of phospho‐Akt, phospho‐eNOS, and Bcl‐2 were decreased, whereas activity of PTEN and caspase‐3 and expression of Bax were increased. Treatment with fasudil normalized these molecular and histologic alterations, and restored erectile function. Insulin treatment showed similar effects to those of fasudil, however, the effects were smaller than fasudil.ConclusionsThis study indicates that up‐regulation of the penile RhoA/ROCK pathway in diabetic rats enhances corporal apoptosis via the PTEN/Akt pathway resulting in ED, which could be prevented by chronic treatment with fasudil. Li WJ, Park K, Paick J‐S, and Kim SW. Chronic treatment with an oral rho‐kinase inhibitor restores erectile function by suppressing corporal apoptosis in diabetic rats.     

Hydroxyl Fasudil,an Inhibitor of Rho Signaling,Improves Erectile Function in Diabetic Rats: A Role for Neuronal ROCK

IntroductionThe pathogenesis of diabetic erectile dysfunction (ED) includes neuropathy, but the molecular basis for neurogenic ED is incompletely understood. The RhoA/ROCK pathway has been implicated in diabetic neuropathy and in ED, but its role in diabetic neurogenic ED is not known.AimsThe aim of this study was to determine whether hydroxyl fasudil, a ROCK inhibitor, affects diabetic neuropathy‐related ED.MethodsType 1 diabetes mellitus was induced in male rats by streptozotocin (75 mg/kg, intraperitoneally). After 8 weeks, diabetic rats were administered hydroxyl fasudil, a selective ROCK inhibitor (10 mg/kg/day, intraperitoneally) or vehicle, for 4 weeks. Age‐matched control, nondiabetic, rats were treated intraperitoneally for 4 weeks with saline. At week 12, after a 2 day washout, neuro‐stimulated erectile function was evaluated. Major pelvic ganglia (MPG) were collected for Western blot analysis of RhoA, ROCK‐1, ROCK‐2, phospho (P)‐AKT (Ser⁴⁷³), and P‐phosphatase and tensin homolog (P‐PTEN) (Ser³⁸⁰/Thr^382/383).Main Outcome MeasuresEffect of ROCK inhibitor hydroxyl fasudil on erectile function and ROCK/P‐AKT/P‐PTEN pathway in the MPG of diabetic rats.ResultsErectile response was significantly (P < 0.05) reduced in diabetic rats compared with nondiabetic rats and was preserved (P < 0.05) in diabetic rats treated with hydroxyl fasudil. In diabetic rats, RhoA and ROCK‐2 protein expressions in MPG were increased (P < 0.05) and remained increased in hydroxyl fasudil‐treated rats. P‐AKT (Ser⁴⁷³) expression was decreased (P < 0.05), whereas P‐PTEN (Ser³⁸⁰/Thr^382/383) expression was increased (P < 0.05) in MPG of diabetic rats compared with nondiabetic rats, and both were reversed (P < 0.05) in diabetic rats treated with hydroxyl fasudil.ConclusionImproved erectile function and restored P‐AKT and P‐PTEN in the MPG with hydroxyl fasudil treatment suggest the role of Rho signaling via PTEN/AKT pathway in neurogenic diabetic ED. Sezen SF, Lagoda G, Musicki B, and Burnett AL. Hydroxyl fasudil, an inhibitor of Rho signaling, improves erectile function in diabetic rats: A role for neuronal ROCK. J Sex Med 2014;11:2164‐2171.     

Apocynin Improves Erectile Function in Diabetic Rats through Regulation of NADPH Oxidase Expression

IntroductionDiabetes is a risk factor for erectile dysfunction (ED). The proposed mechanisms responsible for diabetic ED are associated with an increase in reactive oxygen species (ROS) production, overactivity of RhoA/ROCK signaling pathway and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, as seen in experimental models of diabetic rats.AimThe aim of this study was to investigate whether NADPH oxidase inhibitor apocynin can ameliorate Streptozotocin (STZ)‐induced diabetes‐related ED by reducing the ROS production and inhibiting the activity of RhoA/ROCK signaling pathway.MethodsThe diabetic rats were treated with and without the NADPH oxidase inhibitor apocynin.Main Outcome MeasuresErectile responses were evaluated by determining mean arterial blood pressure (MAP) and intracavernosal pressure (ICP) with electrical stimulation of the cavernous nerve. Levels of mRNA expression were measured by real‐time polymerase chain reaction (RT‐PCR). Levels of protein expression were examined by Western Blot. ROS production was measured by dihydroethidium (DHE) staining and thiobarbituric acid reactive substances assay.ResultsThe ratio of Maximum ICP‐to‐MAP (MaxICP/MAP) was significantly decreased in diabetic ED rats, compared to that of age‐matched control rats (P < 0.05). Apocynin improved erectile function of diabetic rats (P < 0.05). Expression levels of RhoA (cytosol), nNOS and eNOS were reduced, compared to those of control rats (P < 0.05). Apocynin significantly elevated their expression levels in diabetic rats (P < 0.05). Expression levels of ROCK1, RhoA (membrane fraction), p‐MYPT1 and NADPH oxidase subunits p47^phox and p67^phox were increased in diabetic rats when compared to those of control rats (P < 0.05), and it was observed that apocynin significantly reduced their expression levels in diabetic rats (P < 0.05). ROS production was increased in diabetic rats when compared to that of control rats (P < 0.05), the effect of apocynin was a reduction in the ROS production in diabetic rats (P < 0.05).ConclusionNADPH oxidase inhibitor apocynin can ameliorate diabetes‐related ED by reducing the ROS production and inhibiting the activity of RhoA/ROCK signaling pathway. Li M, Zhuan L, Wang T, Rao K, Yang J, Yang J, Quan W, Liu J, and Ye Z. Apocynin improves erectile function in diabetic rats through regulation of NADPH oxidase expression. J Sex Med 2012;9:3041–3050.     

Farnesoid X receptor activation improves erectile function in animal models of metabolic syndrome and diabetes

IntroductionThe farnesoid X receptor (FXR) is critically involved in the regulation of the hepato‐biliary system. Recent data suggest a role for FXR in modulating other metabolic pathways and vascular function.AimTo investigate whether long‐term administration of the selective FXR agonist INT‐747 ameliorates erectile function, we tested it in two animal models of metabolic derangements: a rabbit model of high‐fat diet (HFD)‐induced metabolic syndrome (MetS) and a rat model of streptozotocin (STZ)‐induced type 1 diabetes.MethodsHFD rabbit or STZ rats with or without chronic INT‐747 dosing (10 mg/kg/day for 12 weeks). INT‐747 addition to rabbit penile smooth muscle cells (rpSMCs).Main Outcome MeasureEffects of INT‐747 on metabolic features and erectile function in animal models and clarification of mechanism of action in isolated cells.ResultsINT‐747 dosing normalized visceral adiposity and glucose intolerance in HFD rabbits. INT‐747 increased penile FXR expression and partially restored endothelial nitric oxide synthase and dimethylarginine dimethylaminohydrolase 1 expression as well as impaired nitric oxide (NO)‐dependent relaxation (improved responsiveness to acetylcholine and electrical field stimulation). INT‐747 was also effective in regulating NO downstream events, as shown by increased sodium nitroprusside‐induced relaxation. Because phosphodiesterase type 5 and protein kinase G (PKG) were unaltered by INT‐747, we analyzed the calcium‐sensitizing RhoA/ROCK pathway. HFD increased, and INT‐747 normalized, RhoA membrane translocation/activation. RhoA/ROCK signaling inhibition by INT‐747 was confirmed in rpSMCs by confocal microscopy, MYPT1‐phosphorylation, cytoskeleton remodeling, cell migration, and smooth muscle‐related genes expression. In STZ rats, FXR penile expression was not altered but was significantly upregulated by INT‐747 dosing. In this model, INT‐747 improved penile erection induced by electrical stimulation of cavernous nerve and hypersensitivity to intracavernous injection of a ROCK‐inhibitor, Y‐27632, without improving hyperglycemia.ConclusionIn HFD rabbits, INT‐747 dosing improved glucose sensitivity and MetS‐associated erectile dysfunction, via upregulation of NO transmission and inhibition of RhoA/ROCK pathway. In STZ rats, INT‐747 restored in vivo penile erection and sensitivity to ROCK inhibition, independently of effects on glycemia. Vignozzi L, Morelli A, Filippi S, Comeglio P, Chavalmane AK, Marchetta M, Toce M, Yehiely‐Cohen R, Vannelli GB, Adorini L, and Maggi M. Farnesoid X receptor activation improves erectile function in animal models of metabolic syndrome and diabetes.     

STIM/Orai Inhibition as a Strategy for Alleviating Diabetic Erectile Dysfunction Through Modulation of Rat and Human Penile Tissue Contractility and in vivo Potentiation of Erectile Responses

BackgroundStromal interaction molecule (STIM)/Orai calcium entry system appears to have a role in erectile dysfunction (ED) pathophysiology but its specific contribution to diabetic ED was not elucidated.AimTo evaluate STIM/Orai inhibition on functional alterations associated with diabetic ED in rat and human penile tissues and on in vivo erectile responses in diabetic rats.MethodsRat corpus cavernosum (RCC) strips from nondiabetic (No DM) and streptozotocin-induced diabetic (DM) rats and human penile resistance arteries (HPRA) and corpus cavernosum (HCC) from ED patients undergoing penile prosthesis insertion were functionally evaluated in organ chambers and wire myographs. Erectile function in vivo in rats was assessed by intracavernosal pressure (ICP) responses to cavernous nerve electrical stimulation (CNES). Expression of STIM/Orai elements in HCC was determined by immunofluorescence and immunoblot.Main Outcome MeasuresFunctional responses in RCC, HCC and HPRA and STIM/Orai protein expression in HCC. In vivo erectile responses to CNES.ResultsInhibition of Orai channels with YM-58483 (20 µM) significantly reduced adrenergic contractions in RCC but more effectively in DM. Thromboxane-induced and neurogenic contractions were reduced by STIM/Orai inhibition while defective endothelial, neurogenic and PDE5 inhibitor-induced relaxations were enhanced by YM-58483 (10 µM) in RCC from DM rats. In vivo, YM-58483 caused erections and attenuated diabetes-related impairment of erectile responses. YM-58483 potentiated the effects of PDE5 inhibition. In human tissues, STIM/Orai inhibition depressed adrenergic and thromboxane-induced contractions in ED patients more effectively in those with type 2 diabetes. Diabetes was associated with increased expression of Orai1 and Orai3 in ED patients.Clinical TranslationTargeting STIM/Orai to alleviate diabetes-related functional alterations of penile vascular tissue could improve erectile function and potentiate therapeutic effects of PDE5 inhibitors in diabetic ED.Strengths and LimitationsImproving effects of STIM/Orai inhibition on diabetes-related functional impairment was evidenced in vitro and in vivo in an animal model and validated in human tissues from ED patients. Functional findings were complemented with expression results. Main limitation was low numbers of human experiments due to limited human tissue availability.ConclusionsSTIM/Orai inhibition alleviated alterations of functional responses in vitro and improved erectile responses in vivo in diabetic rats, potentiating the effects of PDE5 inhibition. STIM/Orai inhibition was validated as a target to modulate functional alterations of human penile vascular tissue in diabetic ED where Orai1 and Orai3 channels were upregulated. STIM/Orai inhibition could be a potential therapeutic strategy to overcome poor response to conventional ED therapy in diabetic patients.Sevilleja-Ortiz A, El Assar M, García-Gómez B, et al. STIM/Orai Inhibition as a Strategy for Alleviating Diabetic Erectile Dysfunction Through Modulation of Rat and Human Penile Tissue Contractility and in vivo Potentiation of Erectile Responses. J Sex Med 2022;19:1733–1749.     

Change of erectile function and responsiveness to phosphodiesterase type 5 inhibitors at different stages of streptozotocin-induced diabetes in rats

IntroductionIt has been suggested that risk of erectile dysfunction (ED) increases with duration of diabetes and phosphodiesterase type 5 inhibitors (PDE5I) are not as effective in treatment of diabetes-associated ED. However, few studies have investigated time-dependent change in erectile function during the course of diabetes.AimTo investigate time-dependent change in erectile function and responsiveness to PDE5I in streptozotocin-induced diabetic rats and to understand the pathophysiology of diabetic ED.Main Outcome MeasuresAt 6, 8, 10, 12, and 14 weeks after diabetic induction, erectile function was assessed by cavernous nerve stimulation before and after administration of DA-8159, a novel PDE5I. Penile tissue was assessed for apoptosis with immunohistochemistry. Protein expression of Rho-kinase 2 (ROCK2), myosin phosphatase targeting subunit 1 (MYPT1), and endothelial nitric oxide synthase (eNOS) was evaluated by Western blot.MethodsStreptozotocin was injected into 50 8-week-old male Sprague-Dawley rats, which were then classified into five diabetic groups according to the observation period.ResultsDiabetic rats maintained normal erectile responses until 6 weeks of diabetes. Following 8 weeks, the rats showed lower erectile responses at higher frequencies of nerve stimulation, which were normalized to control by administration of DA-8159. In contrast, erectile responses were significantly decreased in 10-week diabetic rats, and administration of DA-8159 resulted in partial recovery of normal responses. At more than 12 weeks, rats demonstrated severe deterioration of erectile function, which did not fully respond to PDE5I. Corporal apoptosis was significantly increased after 10 weeks. Upregulation of ROCK2 was found at 6 weeks, and was followed by an increase of MYPT1 phosphorylation. Phosphorylation of eNOS showed marked suppression at 6 weeks and remained lower during the experimental period.ConclusionsImpairment of erectile function was followed by decreased responsiveness to PDE5I during the course of diabetes. The RhoA/ROCK pathway played an important role in diabetes-associated ED. Cho SY, Park K, Paick J-S, and Kim SW. Change of erectile function and responsiveness to PDE5 (Type 5 phosphodiesterase) inhibitors at different stages of streptozotocin-induced diabetes in rats.     

Tadalafil Effect on Metabolic Syndrome‐Associated Bladder Alterations: An Experimental Study in a Rabbit Model

IntroductionMetabolic syndrome (MetS) and lower urinary tract symptoms (LUTS) are often associated. Bladder detrusor hyper‐contractility—a major LUTS determinant—is characterized by increased Ras homolog gene family, member A/Rho‐associated protein kinase (RhoA/ROCK) signaling, which is often upregulated in MetS.AimThis study investigated the effects of tadalafil dosing on RhoA/ROCK signaling in bladder, in a rabbit model of high‐fat diet (HFD)‐induced MetS.MethodsAdult male rabbits feeding a HFD for 12 weeks. A subset of HFD animals was treated with tadalafil (2 mg/kg/day, 1 week: the last of the 12 weeks) and compared with HFD and control (feeding a regular diet) rabbits.Main Outcome MeasuresIn vitro contractility studies to evaluate the relaxant effect of the selective ROCK inhibitor, Y‐27632, in carbachol precontracted bladder strips. Evaluation of RhoA activation by its membrane translocation. Immunohistochemistry for ROCK expression has been performed to evaluate ROCK expression in bladder from the different experimental groups. mRNA expression of inflammation, pro‐fibrotic markers by quantitative RT‐PCR has been performed to evaluate the effect of tadalafil on MetS‐induced inflammation and fibrosis within the bladder. The in vitro effect of tadalafil on RhoA/ROCK signaling in bladder smooth muscle cells was evaluated by using chemotaxis assay.ResultsBladder strips from HFD rabbits showed hyper‐responsiveness to Y‐27632, indicating RhoA/ROCK overactivity in HFD bladder compared with matched controls. Accordingly, the fraction of activated (translocated to the membrane) RhoA as well as ROCK expression are increased in HFD bladder. Tadalafil dosing normalized HFD‐induced bladder hypersensitivity to Y‐27632, by reducing RhoA membrane translocation and ROCK overexpression. Tadalafil dosing reduced mRNA expression of inflammatory, pro‐fibrotic, and hypoxia markers. A direct inhibitory effect of tadalafil on RhoA/ROCK signaling in bladder smooth muscle cell was demonstrated by using chemotaxis assay. Pre‐treatment with tadalafil inhibited both basal and PDGF‐induced migration of bladder smooth muscle cells.ConclusionsTadalafil dosing reduced RhoA/ROCK signaling and smooth muscle overactivity in an animal model of MetS‐associated bladder alterations. Our findings suggest a novel mechanism of action of tadalafil in alleviating LUTS in MetS patients. Vignozzi L, Filippi S, Comeglio P, Cellai I, Morelli A, Maneschi E, Sarchielli E, Gacci M, Carini M, Vannelli GB, and Maggi M. Tadalafil effect on metabolic syndrome‐associated bladder alterations: An experimental study in a rabbit model. J Sex Med 2014;11:1159–1172.     

Sex Steroid Receptors in Male Human Bladder: Expression and Biological Function

IntroductionIn male, lower urinary tract symptoms (LUTS) have been associated, beside benign prostatic hyperplasia, to some unexpected comorbidities (hypogonadism, obesity, metabolic syndrome), which are essentially characterized by an unbalance between circulating androgens/estrogens. Within the bladder, LUTS are linked to RhoA/Rho-kinase (ROCK) pathway overactivity.AimTo investigate the effects of changing sex steroids on bladder smooth muscle.MethodsERα, ERβ, GPR30/GPER1 and aromatase mRNA expression was analyzed in male genitourinary tract tissues, and cells isolated from bladder, prostate, and urethra. Estrogen and G1 effect on RhoA/ROCK signaling output like cell migration, gene expression, and cytoskeletal remodeling, and [Ca²⁺]_i was also studied in hB cells. Contractile studies on bladder strips from castrated male rats supplemented with estradiol and testosterone was also performed.Main Outcome MeasuresThe effects of classical (ERα, ERβ) and nonclassical (GPR30/GPER1) estrogen receptor ligands (17β-estradiol and G1, respectively) and androgens on RhoA/ROCK-.mediated cell functions were studied in hB cells. Contractility studies were also performed in bladder strips from castrated male rats supplemented with testosterone or estradiol.ResultsAromatase and sex steroid receptors, including GPR30, were expressed in human bladder and mediates several biological functions. Both 17β-estradiol and G1 activated calcium transients and induced RhoA/ROCK signaling (cell migration, cytoskeleton remodeling and smooth muscle gene expression). RhoA/ROCK inhibitors blunted these effects. Estrogen-, but not androgen-supplementation to castrated rats increased sensitivity to the ROCK inhibitor, Y-27632 in isolated bladder strips. In hB cells, testosterone elicited effects similar to estrogen, which were abrogated by blocking its aromatization through letrozole.ConclusionOur data indicate for the first time that estrogen-more than androgen-receptors up-regulate RhoA/ROCK signaling. Since an altered estrogen/androgen ratio characterizes conditions, such as aging, obesity and metabolic syndrome, often associated to LUTS, we speculate that a relative hyperestrogenism may induce bladder overactivity through the up-regulation of RhoA/ROCK pathway. Chavalmane AK, Comeglio P, Morelli A, Filippi S, Fibbi B, Vignozzi L, Sarchielli E, Marchetta M, Failli P, Sandner P, Saad F, Gacci M, Vannelli GB, and Maggi M. Sex steroid receptors in male human bladder: Expression and biological function.     

Rho Kinase (ROK)‐Related Proteins in Human Cavernous Arteries: An Immunohistochemical and Functional Approach

IntroductionRho kinases (ROKs) cause calcium‐independent modulation of smooth muscle contraction. A significant role for the RhoA/ROK pathway in mediating the contraction of the penile erectile tissue has been suggested. Moreover, it has been postulated that ROK activity might represent a key factor in the pathophysiology of erectile dysfunction. Up until today, little is known on the significance of ROK and related proteins in the control of blood flow in the corpus cavernosum.AimTo investigate by means of immunohistochemistry and organ bath studies the significance of the Rho pathway in human cavernous arteries.Main Outcome MeasuresThe expression of ROK1, ROK2, RhoA, and RhoGDI in human cavernous arteries was investigated by means of immunohistochemistry; myographic studies were conducted in order to characterize the effects of the ROK inhibitor Y27632 on isolated cavernous arteries.MethodsSpecimens of human cavernous arteries were processed for immunohistochemistry for ROK1, ROK2, RhoA, and RhoGDI. Circular penile vascular segments were mounted in a tissue bath and the effects of increasing concentrations of the ROK inhibitor Y27632 on the tension induced by norepinephrine (NE, 1 µM) were investigated.ResultsAlpha‐actin immunoreactive cavernous arterioles also presented abundant staining specific for ROK1, ROK2, RhoA, and RhoGDI in the smooth musculature of the vascular wall. Cumulative addition of Y27632 dose‐dependently reversed the tension induced by NE of isolated arterial segments. Y27632 produced relaxant responses with a reversion of tension of 34.3 ± 11.8% at a concentration of 1 µM.ConclusionThe findings are in support for a role of the Rho/ROK‐mediated signaling in the regulation of muscle tone of human cavernous arteries. Waldkirch ES, Ückert S, Sohn M, Kuczyk MA, and Hedlund P. Rho kinase (Rok)‐related proteins in human cavernous arteries: An immunohistochemical and functional approach. J Sex Med 2012;9:1337–1343.     

The Novel Antioxidant,AC3056 (2,6-di-t-butyl-4-((Dimethyl-4-Methoxyphenylsilyl)Methyloxy)Phenol), Reverses Erectile Dysfunction in Diabetic Rats and Improves NO-mediated Responses in Penile Tissue from Diabetic Men

IntroductionDiabetes is associated with a high incidence of erectile dysfunction (ED) and poor response to standard treatments. Oxidative stress could be relevant in the pathophysiology of diabetic ED.AimTo evaluate the effects of the antioxidant, AC3056 (2,6-di-t-butyl-4-((dimethyl-4-methoxyphenylsilyl)methyloxy)phenol), on diabetic ED.MethodsErectile responses to cavernosal nerve electrical stimulation were determined in streptozotocin-induced diabetic rats. Relaxation of human corpus cavernosal (HCC) tissue and penile resistance arteries (HPRA) from human cavernosal specimens was evaluated in organ chambers and myographs, respectively.Main Outcome MeasuresThe influence of AC3056 on erectile responses, lipid peroxidation, and nitrite plus nitrate serum content, and nuclear factor-κB (NF-κB) expression in penile tissue, in diabetic rats, and on endothelium-dependent and neurogenic relaxation of HCC and HPRA from diabetic patients was determined.ResultsEight weeks of diabetes caused ED in rats that was prevented by oral AC3056 (0.3% w/w in rat chow) when given from the induction of diabetes. AC3056 also prevented the diabetes-induced elevation of serum thiobarbituric acid-reactive substances (TBARS), the reduction of serum nitric oxide (NO) derivatives, and the increase of NF-κB expression. Acute oral administration of AC3056 (450 mg/kg) partially reversed ED in 8-week diabetic rats. Complete reversion of ED was achieved after 3 days of treatment with 0.3% AC3056. This effect remained after 5 weeks of treatment, but it disappeared after withdrawing for 1 week. Erectile function in diabetic rats was inversely related to serum TBARS. AC3056- (30 µM) reversed endothelial dysfunction in diabetic HCC and enhanced endothelium-dependent relaxation in diabetic HPRA and significantly potentiated neurogenic relaxation of both tissues. The reduced cGMP content in HCC from diabetic patients after exposure to acetylcholine (10 µM) was corrected by AC3056 (30 µM).ConclusionsThese results suggest that oxidative stress has a relevant role in pathophysiology of diabetic ED and provide a rationale for the use of antioxidant therapy in the treatment of ED in diabetes. Angulo J, Peiró C, Cuevas P, Gabancho S, Fernández A, González-Corrochano R, La Fuente JM, Baron AD, Chen KS, and Sáenz de Tejada I. The novel antioxidant, AC3056 (2,6-di-t-butyl-4-([dimethyl-4-methoxyphenylsilyl] methyloxy) phenol), reverses erectile dysfunction in diabetic rats and improves NO-mediated responses in penile tissue from diabetic men. J Sex Med 2009;6:373–387.     

Nebivolol Potentiates the Efficacy of PDE5 Inhibitors to Relax Corpus Cavernosum and Penile Arteries from Diabetic Patients by Enhancing the NO/cGMP Pathway

IntroductionThe efficacy of oral pharmacotherapy for erectile dysfunction (ED) (i.e., type 5 phosphodiesterase [PDE5] inhibitors) is significantly reduced in diabetic patients. Nebivolol is a selective β₁‐blocker used for treating hypertension that has been shown to increase the efficacy of sildenafil to reverse ED in diabetic rats.AimTo evaluate the effects of nebivolol on the efficacy of the PDE5 inhibitors, sildenafil, tadalafil, and vardenafil to relax human corpus cavernosum (HCC) and vasodilate human penile resistance arteries (HPRA) from diabetic patients with ED (DMED). The influence of nebivolol on the capacity of these three PDE5 inhibitors to stimulate cyclic guanosine monophosphate (cGMP) production in HCC was also evaluated.MethodsHCC and HPRA were obtained from organ donors without ED (NEND; n = 18) or patients with diabetes undergoing penile prosthesis implantation (DMED; n = 19). Relaxations of HCC strips and HPRA to sildenafil, tadalafil, and vardenafil were evaluated in organ chambers and wire myographs. cGMP content in HCC was determined by ether extraction and quantification by ELISA.Main Outcome MeasuresEffects of nebivolol on PDE5 inhibitor‐induced relaxation of HCC, vasodilation of HPRA and cGMP accumulation in HCC.ResultsTreatment with nebivolol (1 μM) significantly potentiated sildenafil‐, tadalafil‐ and vardenafil‐induced relaxations of HCC and vasodilations of HPRA from both NEND and DMED. Enhancement of relaxant capacity by nebivolol resulted in reversion of the impairment of PDE5 inhibition‐induced responses in DMED and it was accompanied by enhancing the ability of PDE5 inhibitors to increase cGMP in HCC restoring reduced cGMP levels in HCC from DMED.ConclusionsNebivolol potentiated the capacity of PDE5 inhibitors to relax vascular structures of erectile tissue from diabetic patients by enhancing the nitric oxide (NO)/cGMP pathway in these tissues. These effects suggest a potential therapeutic utility of nebivolol as an adjunct to PDE5 inhibitors for the treatment of ED associated with diabetes. Martínez‐Salamanca JI, La Fuente JM, Cardoso J, Fernández A, Cuevas P, Wright HM, and Angulo J. Nebivolol potentiates the efficacy of PDE5 inhibitors to relax corpus cavernosum and penile arteries from diabetic patients by enhancing the NO/cGMP pathway. J Sex Med 2014;11:1182–1192.     

Altered penile vascular reactivity and erection in the Zucker obese-diabetic rat

IntroductionThe combination of the independent risk factors for erectile dysfunction, obesity, hypertension, and diabetes are manifested collectively in a condition known as metabolic syndrome X. However, the exact mechanism(s) by which the combination of these factors contributes to erectile dysfunction have yet to be elucidated.AimWe hypothesized that protein kinase C (PKC) and Rho-kinase enhanced vascular tone and thus contributed to erectile dysfunction in this condition.MethodsErectile function was evaluated by recording voltage-dependent increases in intracavernosal pressure following stimulation of the cavernosal nerve in 16- to 20-week-old lean and obese-diabetic Zucker rats. Cavernosal tissue contractile and relaxation responses were evaluated in vitro when contracted with phenylephrine, endothelin-1 and relaxed by Rho-kinase, PKC inhibitors or sodium nitroprusside. Additionally, cavernosal tissue Rho-kinase, protein kinase, and nitric oxide synthase isoform expression were evaluated by Western blot.ResultsThe voltage-dependent erectile responses were suppressed by >30% in the obese-diabetic Zucker rat. The maximal stress generated by cavernosal tissue from the obese-diabetic was significantly greater than the lean response by greater than 0.8 mN/mm² for both phenylephrine and endothelin stimulation. The PKC inhibitor, chelerythrine, inhibited more than 30% of the phenylephrine-induced and 70% of the endothelin-1-induced contractions. Rho-kinase inhibition, with either Y-27632 or HA-1077, revealed impaired relaxations of nearly 30% in tissue from obese-diabetic animals. Western blot analysis revealed increased protein expression of PKC α and δ and Rho-kinase α and β but no loss for endothelial or neuronal nitric oxide synthase.ConclusionsIn this rodent model both PKC and Rho-kinase signaling elements may contribute to an enhanced vasoconstriction state of the penile smooth muscle that was differentially dependent on the agonist used. The enhanced vasoconstrictive state of the tissue could contribute to the reduced voltage-dependent erectile response in the obese-diabetic Zucker rat. Wingard C, Fulton D, and Husain S. Altered penile vascular reactivity and erection in the Zucker obese-diabetic rat.     

Activated Rho Kinase Mediates Diabetes‐Induced Elevation of Vascular Arginase Activation and Contributes to Impaired Corpora Cavernosa Relaxation: Possible Involvement of p38 MAPK Activation

IntroductionActivated RhoA/Rho kinase (ROCK) has been implicated in diabetes‐induced erectile dysfunction. Earlier studies have demonstrated involvement of ROCK pathway in the activation of arginase in endothelial cells. However, signaling pathways activated by ROCK in the penis remain unclear.AimWe tested whether ROCK and p38 MAPK are involved in the elevation of arginase activity and subsequent impairment of corpora cavernosal (CC) relaxation in diabetes.MethodsEight weeks after streptozotocin‐induced diabetes, vascular functional studies, arginase activity assay, and protein expression of RhoA, ROCK, phospho‐p38 MAPK, p38 MAPK, phospho‐MYPT‐1^Thr850, MYPT‐1 and arginase levels were assessed in CC tissues from nondiabetic wild type (WT), diabetic (D) WT (WT + D), partial ROCK 2^+/? knockout (KO), and ROCK 2^+/? KO + D mice.Main Outcome MeasuresThe expression of RhoA, ROCK 1 and 2, phosphorylation of MYPT‐1^Thr850 and p38 MAPK, arginase activity/expression, endothelial‐ and nitrergic‐dependent relaxation of CC was assayed.ResultsDiabetes significantly reduced maximum relaxation (E_max) to both endothelium‐dependent acetylcholine (WT + D: E_max; 61 ± 4% vs. WT: E_max; 75 ± 2%) and nitrergic nerve stimulation. These effects were associated with increased expression of active RhoA, ROCK 2, phospho‐MYPT‐1^Thr850, phospho‐p38 MAPK, arginase II, and activity of corporal arginase (1.6‐fold) in WT diabetic CC. However, this impairment in CC of WT + D mice was absent in heterozygous ROCK 2^+/? KO + D mice for acetylcholine (E_max: 80 ± 5%) and attenuated for nitrergic nerve‐induced relaxation. CC of ROCK 2^+/? KO + D mice showed much less ROCK activity, did not exhibit p38 MAPK activation, and had reduced arginase activity and arginase II expression. These findings indicate that ROCK 2 mediates diabetes‐induced elevation of arginase activity. Additionally, pretreatment of WT diabetic CC with inhibitors of arginase (ABH) or p38 MAPK (SB203580) partially prevented impairment of ACh‐ and nitrergic nerve‐induced relaxation and elevation of arginase activity.ConclusionROCK 2, p38 MAPK and arginase play key roles in diabetes‐induced impairment of CC relaxation.     

Chronic Administration of Sildenafil Modified the Impaired VEGF System and Improved the Erectile Function in Rats with Diabetic Erectile Dysfunction

IntroductionMen frequently develop diabetic erectile dysfunction (DMED), as a result of endothelial dysfunction. DMED patients often have reduced efficacy with phosphodiesterase type 5 inhibitors therapy.AimTo determine whether chronic sildenafil administration can modify the impaired vascular endothelial growth factor (VEGF) system and improve the erectile function in rats with diabetic erectile dysfunction.MethodsA group of Sprague Dawley rats (n = 30) with DMED were induced by intraperitoneal injection of streptozotocin (40 mg/kg) and screened by subcutaneous injection of Apomorphine (100 mg/kg). They were then exposed to either vehicle or sildenafil (prescribed in our hospital, 5 mg/kg and 10 mg/kg, respectively) for 10 weeks. An additional nondiabetic and age-matched control group (n = 10) was also allocated and given the routine diet for the same period. Assessments were performed to both groups at 36 hours after the last dose of sildenafil. Penile intracavernous pressure (ICP), mean arterial pressure (MAP), penile tissue morphology, immunohistologic analysis, and Western blot analysis of VEGF, VEGFR1, and eNOS were determined.Main Outcome MeasureFunctional, morphological, and proteomical changes on penile structures by the chronic Sildenafil (5 mg/kg and 10 mg/kg, respectively) administration were determined.ResultsA significant increase of ICP, ICP/MAP ratio, and area under the curve were observed in the both groups treated by sildenafil (5 mg/kg and 10 mg/kg, respectively), compared with the DMED rats without receiving Sildenafil. Immunohistochemical staining of their penile tissue showed a decrease in VEGF, VEGFR1, and eNOS staining in the controlled group compared with an improvement in the chronic sildenafil administration group. Western blot analysis demonstrated exactly the same results.ConclusionWe demonstrated that daily sildenafil administration can restore the impaired VEGF system in the penis of DMED rats and progressively improve both erectile function and endothelial function, suggesting a potential general mechanism of improved signaling through the VEGF/eNOS signaling cascade. Liu G, Sun X, Dai Y, Zheng F, Wang D, Huang Y, Bian J, and Deng C. Chronic administration of sildenafil modified the impaired VEGF system and improved the erectile function in rats with diabetic erectile dysfunction.     

Edaravone ameliorates diabetes-induced dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle in the rat

IntroductionDiabetes mellitus (DM) represents a major risk factor for erectile dysfunction (ED). Although the etiology of diabetes-induced ED is multifactorial and still unknown, reactive oxygen species are thought to be one of the key factors.AimThe aim of this article is to investigate whether administration of edaravone, a free radical scavenger, could prevent type 1 diabetes-induced dysfunction of nitric oxide (NO)-induced relaxation in corpus cavernosum smooth muscle in the rat.MethodsSix-week-old male Wistar rats were randomly divided into three groups. One group was treated with citrate-phosphate buffer plus normal saline (group Cont), whereas in the other two groups, diabetes was induced by streptozotocin (50 mg/kg intraperitoneally [i.p.]). Subsequently, the diabetic rats were treated for 4 weeks either with edaravone (10 mg/kg/day, i.p.; group DM + E) or with normal saline (group DM).Main Outcome MeasuresSerum glucose and malondialdehyde levels as well as penile cyclic guanosine monophosphate (cGMP) concentrations were determined, and penile function was estimated by organ bath studies with norepinephrine-mediated contractions and acetylcholine-mediated relaxations. The participation mRNA levels of muscarinic M₃ receptors, neuronal nitrous oxide synthase (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS), and participation protein levels of nNOS, eNOS, phosphorylated nNOS, and phosphorylated eNOS were investigated by quantitative real-time polymerase chain reaction (PCR) and immunoblot analysis, respectively.ResultsTreatment with edaravone prevented partially but significantly the decreased body and penile weight induced by diabetes. Treatment with edaravone significantly improved the increased diabetes-induced malondialdehyde levels, the decreased penile cGMP concentrations, the increased diabetes-induced norepinephrine-mediated contractions, and the decreased acetylcholine-mediated relaxation. Although there were no significant differences in expression levels of mRNAs in nNOS, diabetes-induced upregulation of muscarinic M₃ receptor and iNOS mRNAs as well as diabetes-induced downregulations of eNOS, phosphorylated nNOS, and phosphorylated eNOS were significantly prevented by edaravone.ConclusionsEdaravone decreases the oxidative insult in the penile corpus cavernosum by ameliorating the NO–NOS system and thus preventing partially the developing ED in DM in the rat. Ohmasa F, Saito M, Tsounapi P, Dimitriadis F, Inoue S, Shomori K, Shimizu S, Kinoshita Y, and Satoh K. Edaravone ameliorates diabetes-induced dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle in the rat.