PKC Inhibition Improves Human Penile Vascular Function and the NO/cGMP Pathway in Diabetic Erectile Dysfunction: The Role of NADPH Oxidase.

Erectile dysfunction (ED) is a frequent and difficult-to-treat condition in diabetic men. Protein kinase C (PKC) is involved in diabetes-related vascular and cavernosal alterations. We aimed to evaluate the role of PKC in endothelial dysfunction and NO/cGMP impairment associated with diabetic ED in the human corpus cavernosum (CC) and penile resistance arteries (PRAs) and the potential mechanisms involved. Functional responses were determined in the CC and PRAs in patients with non-diabetic ED and diabetic ED undergoing penile prosthesis insertion. PKC activator 12,13-phorbol-dibutyrate (PDBu) impaired endothelial relaxations and cGMP generation in response to acetylcholine in the CC from non-diabetic ED. PDBu also impaired responses to a PDE5 inhibitor, sildenafil, in non-diabetic ED patients. Conversely, a PKC inhibitor, GF109203X, improved endothelial, neurogenic, and PDE5-inhibitor-induced relaxations and cGMP generation only in the CC in diabetic ED patients. Endothelial and PDE5-inhibitor-induced vasodilations of PRAs were potentiated only in diabetes. Improvements in endothelial function in diabetes were also achieved with a specific inhibitor of the PKCß2 isoform or an NADPH-oxidase inhibitor, apocynin, which prevented PDBu-induced impairment in non-diabetic patients. PKC inhibition counteracted NO/cGMP impairment and endothelial dysfunction in diabetes-related ED, potentially improving response to PDE5 inhibition.

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.

BACKGROUND: Stromal 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. AIM: To 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. METHODS: Rat 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 MEASURES: Functional responses in RCC, HCC and HPRA and STIM/Orai protein expression in HCC. In vivo erectile responses to CNES. RESULTS: Inhibition 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 TRANSLATION: Targeting 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 LIMITATIONS: Improving 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. CONCLUSIONS: STIM/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.

Enhanced Contribution of Orai Channels to Contractility of Human Penile Smooth Muscle in Erectile Dysfunction.

BACKGROUND: Store-operated calcium entry and its key players, stromal interaction molecule (STIM) and Orai calcium channels, have been proposed as emergent therapeutic targets in cardiovascular pathophysiology. We hypothesize alteration of STIM/Orai signaling in erectile dysfunction (ED). AIM: To evaluate the contribution of STIM/Orai to human penile tissue contraction and to analyze the influence of ED on STIM/Orai signaling at functional and expression levels in human penile vascular tissues. METHODS: Human penile resistance arteries (HPRA) and human corpus cavernosum (HCC) were dissected from cavernosal specimens from 30 organ donors without history of ED (No ED) and from 48 patients with ED undergoing penile prosthesis insertion and functionally evaluated in wire myographs and organ chambers, respectively. Expression of STIM-1, Orai1, and Orai3 in HCC was localized and quantified by immunofluorescence. MAIN OUTCOME MEASURES: The main outcome measures are functional responses in HCC and HPRA and STIM/Orai channel protein expression in human cavernosal tissue. RESULTS: Inhibition of Orai channels with YM-58483 (20 µM) significantly reduced norepinephrine-induced contractions in both HCC and HPRA from either No ED or ED subjects, but the effects were more marked in ED (-20.1 ± 5.9% vs -45.5 ± 13.2% and -15.9 ± 4.0% vs -31.4 ± 6.9% reduction in Emax to norepinephrine in HCC and HPRA, respectively). Thromboxane-induced contractions were reduced and neurogenic contractile and relaxant responses modulated by Orai inhibition in penile tissues from patients with ED. In fact, addition of YM-58483 concentration dependently relaxed precontracted HPRA and HCC. These relaxations were significantly more pronounced in tissues from patients with ED (EC50 7.5 vs 1.3 µM and 10.5 vs 1.3 µM, for HCC and HPRA, respectively). All HCC specimens displayed expression of STIM-1, Orai1, and Orai3. Significantly increased expression of Orai1 and Orai3 but not STIM-1 was observed in patients with ED. CLINICAL TRANSLATION: Inhibition of enhanced Orai activity in human penile vascular tissue could facilitate erectile responses, alleviating ED. STRENGTHS AND LIMITATIONS: Enhanced STIM/Orai activity contribution to penile smooth muscle tone in ED is demonstrated at functional and structural levels in human tissues from a representative sample of patients with ED and in comparison with healthy tissue. We cannot differentiate the specific contribution of risk factors associated with ED to hyperactivity of the Orai system. CONCLUSIONS: Orai channels significantly contribute to human penile smooth muscle contraction. Orai contribution to penile smooth muscle tone is functionally enhanced in ED accompanied by increased expression of Orai channels in cavernosal tissue. Orai inhibition could be a potential therapeutic strategy to reduce penile smooth muscle contraction in ED. Sevilleja-Ortiz A, El Assar M, García-Rojo E, et al. Enhanced Contribution of Orai Channels to Contractility of Human Penile Smooth Muscle in Erectile Dysfunction. J Sex Med 2020;17:881-891.

A novel approach for classifying protruding ears is easy to calculate and implement in daily clinical practice.

AIM: Protruding ears are a prevalent deformity, with a reported incidence of 5% in the paediatric population, but it lacks a simple digital classification. The aim of this study was to find a parameter that would objectively allow the photographic classification of protrusion, by comparing frontal facial images of patients with protruding ears with aged-matched controls. METHODS: This prospective cohort study compared the frontal facial images of 105 patients with protruding ears with 112 aged-matched controls without protruding ears. A rectangle was drawn on the image for each ear, encompassing its full visible anatomy. The width of each rectangle was divided by its height to create an index. The mean value of both ears was defined as the Frontal Aesthetics Translation Index for Measurement of Amplitude of the Ears (FATIMAE). RESULTS: The calculated values for group with protruding ears were significantly higher than for the controls. No gender differences were found. However, the FATIMAE values decreased with age, establishing different classification criteria for separate age groups. CONCLUSION: The FATIMAE is easy to calculate and implement in daily clinical practice and establishes a practical approach for classification of protrusion, as well as for referral criteria for a specialised surgical consultation.

α1A-Adrenergic Receptor Antagonism Improves Erectile and Cavernosal Responses in Rats With Cavernous Nerve Injury and Enhances Neurogenic Responses in Human Corpus Cavernosum From Patients With Erectile Dysfunction Secondary to Radical Prostatectomy.

INTRODUCTION: Cavernous nerve injury (CNI) in rats and radical prostatectomy (RP) in men result in loss of nitrergic function and increased adrenergic-neurogenic contractions of cavernosal tissue. AIM: To evaluate the modulation of the α-adrenergic system as a strategy to relieve erectile dysfunction (ED) and functional cavernosal alterations induced by CNI. METHODS: A non-selective α-blocker (phentolamine 1 mg/kg daily), a selective α1A-blocker (silodosin [SILOD] 0.1 mg/kg daily), or vehicle was orally administered for 4 weeks after bilateral crush CNI (BCNI). Erectile and neurogenic responses of the corpus cavernosum (CC) were evaluated. The acute effects of SILOD also were evaluated in vivo (0.03 mg/kg intravenously) and ex vivo (10 nmol/L). The effects of SILOD and tadalafil (TAD) on nitrergic relaxations were determined in human CC from patients with ED with a vascular etiology or ED secondary to RP. MAIN OUTCOME MEASURES: Erectile responses in vivo in rats and neurogenic contractions and relaxations of rat and human CC. RESULTS: Long-term treatment with SILOD significantly improved erectile responses and allowed for the potentiation of erectile responses by acute treatment with TAD (0.3 mg/kg intravenously) in rats with BCNI. SILOD partly recovered nitrergic relaxations and normalized neurogenic contractions in CC from rats with BCNI. Long-term treatment with SILOD partly prevented BCNI-induced decreases in neuronal nitric oxide synthase expression. Acute administration of SILOD (0.03 mg/kg intravenously) improved erectile responses in vivo and potentiated nitrergic relaxation and decreased neurogenic contractions ex vivo in CC from rats with BCNI. In human CC from patients with ED with a vascular etiology, TAD (30 nmol/L), SILOD (10 nmol/L), or their combination increased nitrergic relaxations. Potentiation by TAD was lost in human CC from patients with ED after RP but was recovered after co-treatment with SILOD. CONCLUSION: α-Adrenergic modulation, especially selective α1A-blockade, improves erectile and cavernosal functions after BCNI. Modulation of the adrenergic system, mainly in combination strategies, could have a role in the management of ED after RP.

Nitrergic function is lost but endothelial function is preserved in the corpus cavernosum and penile resistance arteries of men after radical prostatectomy.

INTRODUCTION: Radical prostatectomy (RP) frequently results in erectile dysfunction (ED). It has been hypothesized that alterations of cavernosal tissue subsequent to RP contribute to ED but functional evaluation of the impact of RP on human erectile structures is lacking. AIM: This study aims to evaluate endothelial function of human corpus cavernosum (HCC) and human penile resistance arteries (HPRA) and neurogenic responses of HCC from patients with ED secondary to RP (ED-RP). METHODS: HCC strips and HPRA were obtained from organ donors without history of ED (No-ED) and patients with ED who were segregated depending on ED etiology: ED-RP or vasculogenic (ED-VASC). Functional evaluation of HCC and HPRA was performed in organ chambers and wire myographs, respectively. Histological evaluation of cavernosal tissue consisted of trichrome staining for fibrosis quantification and TUNEL assay for determination of apoptosis. MAIN OUTCOME MEASURES: Endothelium-dependent and endothelium-independent relaxation, electrical field stimulation (EFS)-induced neurogenic contraction and relaxation, and cavernosal fibrosis and apoptosis. RESULTS: Endothelium-dependent relaxations were significantly impaired in HCC and HPRA from ED-VASC patients while these responses in ED-PR patients were not different to No-ED. Similarly, sildenafil-induced relaxations were reduced in HCC and HPRA from ED-VASC but were preserved in ED-RP. Adrenergic contractions induced by EFS in HCC were potentiated in both ED-RP and ED-VASC. EFS-induced nitrergic relaxation was significantly reduced in HCC from ED-VASC but was almost abolished in ED-RP. Fibrous tissue content and cavernosal apoptosis in HCC from ED-RP were not significantly different from No-ED. CONCLUSIONS: Endothelial function and cavernosal sensitivity to phosphodiesterase type 5 inhibitors are preserved in erectile tissue from ED-RP while a marked imbalance in neurogenic modulation of cavernosal tone favoring adrenergic contractile responses over nitrergic relaxation is manifested. Fibrotic and apoptotic processes in cavernosal tissue are not specifically associated to ED-RP. These evidences could help to retarget therapeutic strategies in the management of ED after RP.

Nebivolol potentiates the efficacy of PDE5 inhibitors to relax corpus cavernosum and penile arteries from diabetic patients by enhancing the NO/cGMP pathway.

INTRODUCTION: The 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 ß1-blocker used for treatinghy pertension that has been shown to increase the efficacy of sildenafil to reverse ED in diabetic rats. AIM: To 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. METHODS: HCC 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 MEASURES: Effects of nebivolol on PDE5 inhibitor-induced relaxation of HCC, vasodilation ofHPRA and cGMP accumulation in HCC. RESULTS: Treatment 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 levelsin HCC from DMED. CONCLUSIONS: Nebivolol 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.

LDL-c/HDL-c Ratio and NADPH-Oxidase-2-Derived Oxidative Stress as Main Determinants of Microvascular Endothelial Function in Morbidly Obese Subjects.

The identification of obese subjects at higher risk for cardiovascular disease (CVD) is required. We aimed to characterize determinants of endothelial dysfunction, the initial step to CVD, in small omental arteries of visceral fat from obese subjects. The influences of analytical parameters and vascular oxidative stress mediated by NADPH-oxidase-2 (NOX2) on endothelial function were determined. Specimens were obtained from 51 obese subjects undergoing bariatric surgery and 14 non-obese subjects undergoing abdominal surgery. Obese subjects displayed reduced endothelial vasodilation to bradykinin (BK). Endothelial vasodilation (pEC50 for BK) among obese subjects was significantly and negatively associated with low-density lipoprotein cholesterol (LDL-c)/high-density lipoprotein cholesterol (HDL-c) ratio (r = -0.510, p = 0.0001) in both women and men, while other metabolic parameters and comorbidities failed to predict endothelial function. The vascular expression of NOX2 was upregulated in obese subjects and was related to decreased endothelial vasodilation (r = -0.529, p = 0.0006, n = 38) and increased oxidative stress (r = 0.783, p = 0.0044, n = 11) in arterial segments. High LDL-c/HDL-c (>2) and high NOX2 (above median) were independently associated with reduced endothelial function, but the presence of both conditions was related to a further impairment. Concomitant elevated LDL-c/HDL-c ratio and high vascular expression of NOX2 would exacerbate endothelial impairment in obesity and could reveal a deleterious profile for cardiovascular outcomes among obese subjects.

Tadalafil enhances the inhibitory effects of tamsulosin on neurogenic contractions of human prostate and bladder neck.

INTRODUCTION: Lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH-LUTSs) may be associated with erectile dysfunction (ED). Phosphodiesterase type 5 (PDE5) inhibitors used for treating ED have shown clinical benefit in patients with LUTS but their actions in human LUT tissues are not well defined. AIM: To determine the effects of the long-acting PDE5 inhibitor, tadalafil, on smooth muscle tone in human prostate and bladder neck as well as to evaluate the influence of tadalafil on the efficacy of the α-adrenergic receptor antagonist, tamsulosin, in inhibiting contractile responses in these tissues. METHODS: Strips of human peripheral prostate (HPP), human internal prostate (HIP), and human bladder neck (HBN) were obtained from organ donors and patients with BPH. The strips were then disposed in organ baths to evaluate nitric oxide/cyclic guanosine monophosphate (cGMP)-mediated relaxation and cGMP kinetics in HPP and HIP, and electrical field stimulation (EFS)-induced neurogenic contractions in HPP and HBN. MAIN OUTCOME MEASURES: Tadalafil-induced effects on sodium nitroprusside (SNP)-induced relaxation and cGMP accumulation in HPP and HIP and influence of tadalafil and tamsulosin on EFS-induced contractions of HPP and HBN. RESULTS: SNP-induced relaxation of HPP and HIP was significantly potentiated by tadalafil (30-60 nM). SNP-induced cGMP accumulation in HPP and HIP was enhanced by tadalafil (30-60 nM), but significant difference was only obtained in HPP. EFS-induced contractions sensitive to tetrodotoxin in HPP were significantly inhibited by tadalafil (30 nM) but not by tamsulosin (0.01-100 nM) or vehicle. Further inhibition of neurogenic responses in HPP was achieved by combining tadalafil and tamsulosin treatments. Tamsulosin, but not tadalafil, significantly reduced EFS-induced contractions in HBN, but the coadministration of both therapies resulted in additional inhibition of contractions. CONCLUSIONS: While tadalafil enhances cGMP accumulation and potentiates prostate relaxation, tadalafil combined with tamsulosin results in enhanced inhibition of neurogenic contractions of HPP and HBN.

Early manifestation of aging-related vascular dysfunction in human penile vasculature-A potential explanation for the role of erectile dysfunction as a harbinger of systemic vascular disease.

Advanced age is related to functional alterations of human vasculature, but erectile dysfunction precedes systemic manifestations of vascular disease. The current study aimed to simultaneously evaluate the influence of aging on vascular function (relaxation and contraction responses) in systemic human vascular territories: aorta (HA) and resistance mesenteric arteries (HMA) and human corpus cavernosum (HCC) and penile resistance arteries (HPRA). Associations of oxidative stress and inflammation circulating biomarkers with age and functional responses were also determined. Vascular specimens were obtained from 76 organ donors (age range 18-87). Four age-groups were established: < 40, 40-55, 56-65 and > 65 years old. Increasing age was associated with a decline in endothelium-dependent relaxation induced by BK in HMA (r = -0.597, p = 0.0001), or by ACh in HCC (r = -0.505, p = 0.0022), and HPRA (r = -0.601, p = 0.0012). Significant impairment was detected at > 65 years old in HMA but earlier in penile vasculature (> 55 years old). Age-related reduction to H2O2-vasodilatory response started before in HCC (56-65 years old) than in HA (> 65 years old). In contrast to relaxation responses, aging-related hypercontractility to adrenergic stimulation was homogeneous: contractions significantly increased in subjects > 55 years old in all tested vessels. Although not significantly age related, circulating levels of ADMA (r = -0.681, p = 0.0052) and TNF-α (r = -0.537, p = 0.0385) were negatively correlated with endothelial vasodilation in HMA but not in HCC or HPRA. Penile vasculature exhibits an early impairment of endothelium-dependent and H2O2-induced vasodilations when compared to mesenteric microcirculation and aorta. Therefore, functional susceptibility of penile vasculature to the aging process may account for anticipation of erectile dysfunction to systemic manifestations of vascular disease.

Ageing-induced hypercontractility is related to functional enhancement of STIM/Orai and upregulation of Orai 3 in rat and human penile tissue.

The role of STIM/Orai calcium entry system on vascular ageing has not been elucidated. We aimed to evaluate the influence of ageing on STIM/Orai signalling and its role on ageing-induced alterations of contractile function in rat corpus cavernosum (RCC) and human penile resistance arteries (HPRA) and corpus cavernosum (HCC). RCC was obtained from 3 months-old and 20 months-old animals. HPRA and HCC were obtained from organ donors of varied ages without history of erectile dysfunction. Aging was associated with enhanced norepinephrine (NE)- and thromboxane analogue (U46619)-induced contractions in RCC which were significantly inhibited by the STIM/Orai inhibitor, YM-58483 (20 µM). Other STIM/Orai inhibitor, 2-aminoethyldiphenylborate also reduced NE-induced contractions in RCC from aged rats. YM-58483 significantly reduced neurogenic contractions and potentiated neurogenic relaxations in RCC from aged rats. In HCC and HPRA, NE-induced contractions were significantly enhanced in older subjects (>65 years-old) but YM-58483 completely reversed ageing-related hypercontractility. Ageing did not modify STIM-1 and Orai1 protein expressions but Orai3 was significantly overexpressed in cavernosal tissue from old rats and older subjects. Contribution of STIM/Orai to cavernosal contraction increases with ageing together with increased expression of Orai3. Orai inhibition could be a potential therapeutic strategy to reduce ageing-related impact on vascular/erectile function.

Efficacy of a proprioceptive exercise program in patients with nonspecific neck pain: a randomized controlled trial.

BACKGROUND: Nonspecific neck pain is associated with chronic pain, disability, reduced cervical mobility, postural control disorders and impaired proprioceptive control. AIM: The aim of this study was to compare the effectiveness of two therapeutic exercise programs (i.e. cervical proprioception and cervical mobility) in reducing pain and disability in individuals with nonspecific neck pain. We further aimed to compare the effectiveness of the two treatments in improving pressure pain threshold, cervical range of motion and head repositioning accuracy. DESIGN: This study was designed as a randomized controlled trial. SETTING: This study took place in a private rehabilitation clinic. POPULATION: Forty-two participants diagnosed with nonspecific neck pain, aged 18-65 years, were randomized to a cervical mobility group (N.=22) or a proprioception group (N.=20). METHODS: The cervical mobility group combined a passive treatment and active mobility exercises, whereas the Proprioception group combined a passive treatment and proprioceptive exercises. Pain intensity, disability, pressure pain threshold, range of motion, and head repositioning accuracy were assessed at baseline and after 10 sessions. RESULTS: Pain intensity and disability significantly improved for both interventions (p<0.01), but such improvement was greater for pain intensity in the proprioception group than in the cervical mobility group (P<0.01). Pressure pain threshold, range of motion and head repositioning accuracy improved only in the proprioception group (P<0.01). CONCLUSIONS: A program based on cervical proprioception exercises demonstrated to improve pain, disability, pressure pain threshold, range of motion and head repositioning accuracy in patients with nonspecific neck pain. However, a program based on cervical mobility exercises only showed to improve pain intensity and disability, while such improvement was not clinically relevant. CLINICAL REHABILITATION IMPACT: The proprioceptive exercise program may be considered as the treatment of choice in patients with nonspecific neck pain.

Diabetes exacerbates the functional deficiency of NO/cGMP pathway associated with erectile dysfunction in human corpus cavernosum and penile arteries.

INTRODUCTION: Diabetic men with erectile dysfunction (ED) are less responsive to therapy with type 5 phosphodiesterase (PDE5) inhibitors. Although an impairment of the nitric oxide (NO)/cyclic guanosin-monophosphate (cGMP) pathway has been shown in diabetic ED vs. non-diabetic ED, the functionality of NO/cGMP pathway in non-diabetic and diabetic ED patients with respect to non-ED patients has not been established. AIM: The aim of this study is to evaluate the function of NO/cGMP signalling in human erectile tissues from ED patients exploring the added impact of diabetes. METHODS: Corpus cavernosum strips (human corpus cavernosum [HCC]) and penile resistance arteries (HPRA) were collected from penile specimens from organ donors (OD) and from diabetic and non-diabetic men with ED undergoing penile prosthesis implantation. MAIN OUTCOME MEASURES: Relaxations to acetylcholine, electrical field stimulation, sodium nitroprusside, and sildenafil were evaluated in phenylephrine-contracted HCC and norepinephrine-contracted HPRA. cGMP content in HCC was also determined. RESULTS: The impairment of endothelium-dependent relaxation in HCC and HPRA from ED patients was exacerbated by diabetes (E(max) 76.1, 62.9, and 49.3% in HCC and 73.1, 59.8, and 46.0% in HPRA from OD, non-diabetic and diabetic ED, respectively). Hypertension, hypercholesterolemia, or aging did not exert a further impairment of endothelial relaxation among ED patients. Diabetes also causes a further impairment of neurogenic relaxation in HCC and HPRA. The basal and stimulated content of cGMP in HCC was significantly decreased in patients with ED, but specially reduced in diabetic patients. Diabetes clearly impaired PDE5 inhibitor-induced vasodilation of HPRA from ED patients. CONCLUSIONS: ED is related to impaired vasodilation, reduced relaxant capacity, and diminished cGMP content in penile tissue. These alterations are more severe in diabetes and accompany reduced relaxant efficacy of PDE5 inhibition. Thus, an exacerbated reduction of nitric oxide/cGMP signaling could be responsible for ED in diabetic men and would explain their reduced response to treatment.

Nebivolol dilates human penile arteries and reverses erectile dysfunction in diabetic rats through enhancement of nitric oxide signaling.

INTRODUCTION: Traditional beta-blockers have sometimes been associated with erectile dysfunction (ED). Nebivolol is a cardioselective ß(1)-adrenoceptor antagonist that promotes vasodilation through a nitric oxide (NO)-dependent mechanism. AIM: We 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. METHODS: Erectile 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 MEASURES: The effects of nebivolol on erectile function and dysfunction and on NO/cGMP-mediated responses. RESULTS: Treatment 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. CONCLUSIONS: Nebivolol 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.

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 ß-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.

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.

INTRODUCTION: Diabetes 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. AIM: To evaluate the effects of the antioxidant, AC3056 (2,6-di-t-butyl-4-((dimethyl-4-methoxyphenylsilyl)methyloxy)phenol), on diabetic ED. METHODS: Erectile 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 MEASURES: The influence of AC3056 on erectile responses, lipid peroxidation, and nitrite plus nitrate serum content, and nuclear factor-kappaB (NF-kappaB) expression in penile tissue, in diabetic rats, and on endothelium-dependent and neurogenic relaxation of HCC and HPRA from diabetic patients was determined. RESULTS: Eight 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-kappaB 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 microM) 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 microM) was corrected by AC3056 (30 microM). CONCLUSIONS: These 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.

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 ß-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.

Short-term pharmacological activation of Nrf2 ameliorates vascular dysfunction in aged rats and in pathological human vasculature. A potential target for therapeutic intervention.

Oxidative stress contributes to endothelial dysfunction, a key step in cardiovascular disease development. Ageing-related vascular dysfunction involves defective antioxidant response. Nuclear factor erythroid 2-like-2 (Nrf2), orchestrates cellular response to oxidative stress. We evaluated the impact of Nrf2-activation on endothelium-dependent and H2O2-mediated vasodilations in: aorta (RA), mesenteric artery (RMA), coronary artery (RCA) and corpus cavernosum (RCC) from ageing rats and in human penile arteries (HPRA) and corpus cavernosum (HCC) from erectile dysfunction (ED) patients. Relaxant responses were evaluated in organ chambers and wire myographs. Nrf2 content and heme oxygenase-1 (HO-1) were determined by ELISA. Superoxide and Nrf2 were detected by immunofluorescence. Pharmacological activation of Nrf2 with sulforaphane (SFN) improved NO- and endothelium-derived hyperpolarizing factor-mediated endothelium-dependent vasodilation and H2O2-induced relaxation in vascular beds from aging rats. SFN-induced effects were associated with increased Nrf2 (RMA, RCA) and reduced superoxide detection in RCA. Improvement of vascular function was confirmed in HPRA and HCC from ED patients and mimicked by another Nrf2 activator, oltipraz. Nrf2 increase and superoxide reduction together with HO-1 increase by Nrf2 activation was evidenced in HCC from ED patients. PDE5 inhibitor-induced relaxations of HPRA and HCC from ED patients were enhanced by SFN. Nrf2 short-term pharmacological activation attenuates age-related impairment of endothelium-dependent and reactive oxygen species (ROS)-induced vasodilation in different rat and human vascular territories by upregulation of Nrf2-related signaling and decreased oxidative stress. In ED patients target tissues, Nrf2 potentiates the functional effect of ED conventional pharmacological therapy suggesting potential therapeutic implication.

Regulation of human penile smooth muscle tone by prostanoid receptors.

We have characterized the prostanoid receptors involved in the regulation of human penile arterial and trabecular smooth muscle tone. Arachidonic acid induced relaxation of human corpus cavernosum strips (HCCS) that was blocked by the cyclo-oxygenase inhibitor, indomethacin, and augmented by the thromboxane receptor (TP) antagonist, SQ29548, suggesting that endogenous production of prostanoids regulates penile smooth muscle tone. TP-receptors mediate contraction of HCCS and penile resistance arteries (HPRA), since the agonist of these receptors, U46619, potently contracted HCCS (EC50 8.3+/-2.8 nM) and HPRA (EC50 6.2+/-2.2 nM), and the contractions produced by prostaglandin F(2alpha) at high concentrations (EC50 6460+/-3220 nM in HCCS and 8900+/-6700 nM in HPRA) were inhibited by the selective TP-receptor antagonist, SQ29548 (0.02 microM). EP-receptors are responsible for prostanoid-induced relaxant effects in HCCS because only prostaglandin E1 (PGE1), prostaglandin E2 and the EP2/EP4-receptor agonist, butaprost, produced consistent relaxation of this tissue (EC50 93.8+/-31.5, 16.3+/-3.8 and 1820+/-1284 nM, respectively). In HPRA, both prostacyclin and PGE1 (EC50 60.1+/-18.4 and 109.0+/-30.9 nM, respectively) as well as the selective IP receptor agonist, cicaprost, and butaprost (EC50 25.2+/-15.2 and 7050+/-6020 nM, respectively) caused relaxation, suggesting co-existence of IP- and EP-receptors (EP2 and/or EP4). In summary, endogenous production of prostanoids may regulate penile smooth muscle contractility by way of specific receptors. TP-receptors mediate contraction in HCCS and HPRA, while the relaxant effects of prostanoids are mediated by EP2- and/or EP4-receptors in HCCS and by EP- and IP-receptors in HPRA.

Stimulation of large-conductance calcium-activated potassium channels inhibits neurogenic contraction of human bladder from patients with urinary symptoms and reverses acetic acid-induced bladder hyperactivity in rats.

We have analysed the effects of large-conductance calcium-activated potassium channel (BK) stimulation on neurogenic and myogenic contraction of human bladder from healthy subjects and patients with urinary symptoms and evaluated the efficacy of activating BK to relief bladder hyperactivity in rats. Bladder specimens were obtained from organ donors and from men with benign prostatic hyperplasia (BPH). Contractions elicited by electrical field stimulation (EFS) and carbachol (CCh) were evaluated in isolated bladder strips. in vivo cystometric recordings were obtained in anesthetized rats under control and acetic acid-induced hyperactive conditions. Neurogenic contractions of human bladder were potentiated by blockade of BK and small-conductance calcium-activated potassium channels (SK) but were unaffected by the blockade of intermediate calcium-activated potassium channels (IK). EFS-induced contractions were inhibited by BK stimulation with NS-8 or NS1619 or by SK/IK stimulation with NS309 (3µM). CCh-induced contractions were not modified by blockade or stimulation of BK, IK or SK. The anti-cholinergic agent, oxybutynin (0.3µM) inhibited either neurogenic or CCh-induced contractions. Neurogenic contractions of bladders from BPH patients were less sensitive to BK inhibition and more sensitive to BK activation than healthy bladders. The BK activator, NS-8 (5mg/kg; i.v.), reversed bladder hyperactivity induced by acetic acid in rats, while oxybutynin was ineffective. NS-8 did not significantly impact blood pressure or heart rate. BK stimulation specifically inhibits neurogenic contractions in patients with urinary symptoms and relieves bladder hyperactivity in vivo without compromising bladder contractile capacity or cardiovascular safety, supporting its potential therapeutic use for relieving bladder overactivity.