Effect of hydrogen sulphide‐donating sildenafil (ACS6) on erectile function and oxidative stress in rabbit isolated corpus cavernosum and in hypertensive rats

OBJECTIVE

To study the effect of the H₂S‐donating derivative of sildenafil (ACS6) compared to sildenafil citrate and sodium hydrosulphide (NaHS) on relaxation, superoxide formation and NADPH oxidase and type 5 phosphodiesterase (PDE5) expression in isolated rabbit cavernosal tissue and smooth muscle cells (CSMCs), and in vivo on indices of oxidative stress induced with buthionine sulphoximine (BSO).

MATERIALS AND METHODS

Relaxation was studied in an organ bath in response to carbachol and after incubation with interleukin‐1β for 12 h. CSMCs were incubated with tumour‐necrosis factor‐α or the thromboxane A₂ (TXA₂) analogue, U46619, with or with no sildenafil citrate, ACS6 or NaHS for 16 h. Superoxide formation and the expression of p47^phox (an active subunit of the NADPH oxidase complex) and PDE5 protein was then assessed using Western blotting. Rats were also treated with BSO (with or with no sildenafil citrate or ACS6) for 7 days; cavernosal cGMP, cAMP, glutathionine and plasma TXA₂ and 8‐isoprostane F_2α was measured by enzyme‐linked immunosorbent assay.

RESULTS

ACS6 and sildenafil citrate relaxed cavernosal smooth muscle equipotently; NaHS alone had little effect at up to 100 µm . The formation of superoxide and expression of p47^phox and PDE5 was reduced by ACS6, sildenafil citrate and NaHS (order of potency: ACS6 > sildenafil citrate > NaHS). The effects of ACS6 were blocked by inhibitors of protein kinase A (PKA) and PKG. In rats treated with BSO, both ASC6 and sildenafil citrate reduced the increased plasma levels of TXA₂ and 8‐isoprostane F_2α but increased cGMP, cAMP and glutathionine levels in corpus cavernosum.

CONCLUSIONS

By virtue of a dual action on PKA and PKG activation, ACS6 not only promotes erection, acutely, but might also have a long‐term beneficial effect through inhibition of oxidative stress and downregulation of PDE5.     

The administration of folic acid improves erectile function and reduces intracavernosal oxidative stress in the diabetic rabbit

OBJECTIVE

To test the possibility that folic acid (FA) may be a means of treating erectile dysfunction (ED) in diabetes mellitus (DM), by studying the effect of FA administration to DM rabbits on cavernosal function and intrapenile oxidative stress.

MATERIALS AND METHODS

To investigate the effect of administering FA to DM rabbits on erectile function and oxidative stress the formation of superoxide (O₂^‐), 8‐isoprostane F_2α (8‐IPF_2α) and prostacyclin (as 6‐keto‐PGF_1α) were assessed, as well as carbachol‐ and electrical field stimulated (EFS) relaxation and p47^phox content (active component of NADPH oxidase complex). Non‐ketotic DM was induced in New Zealand rabbits with alloxan and FA administered orally daily for 1 month. Rabbits were killed, penises excised and segments prepared. These were mounted in an organ bath and relaxation elicited with carbachol or EFS. O₂^‐ release was measured spectrophotometrically, p47^phox expression by Western blotting and 8‐IPF_2α and 6‐keto‐PGF_1α formation by enzyme‐linked immunosorbant assay. Blood was collected for measurement of homocysteine, red blood cell (RBC) folate and glucose.

RESULTS

In cavernosal tissue from DM rabbits, carbachol‐and EFS‐induced relaxation was significantly impaired compared with the untreated controls. O₂^‐ release, p47^phox expression and 8‐IPF_2α formation were all enhanced and 6‐keto‐PGF_1α formation reduced compared with the controls. All these effects were reversed by FA. Plasma total homocysteine was reduced and RBC folate elevated.

CONCLUSIONS

The administration of FA may constitute a strategy for reducing ED in patients with DM.     

Sildenafil inhibits the formation of superoxide and the expression of gp47 NAD[P]H oxidase induced by the thromboxane A2 mimetic, U46619, in corpus cavernosal smooth muscle cells

OBJECTIVE: To assess the effect of sildenafil on superoxide formation and p47(phox) (the active subunit of NADPH oxidase) expression in cultured corpus cavernosal smooth muscle cells (CVSMCs). MATERIALS AND METHODS: CVSMCs derived from rabbit penis were incubated with U46619 (thromboxane A2 analogue) with or without sildenafil for 1 or 16 h at 37 degrees C. Superoxide dismutase-inhibitable superoxide formation was assessed using the reduction of ferricytochrome c measured spectrophotometrically, and gp47(phox) assessed using Western blot analysis. The role of NAD[P]H oxidase and cGMP was further studied by using specific inhibitors of each. RESULTS: Superoxide formation was significantly greater in cells incubated with U46619 after 1 and 16 h incubation than in controls, an effect blocked by NADP(H) oxidase inhibitors. These effects of U46619 were inhibited by sildenafil (1 and 10 nmol/L), which in turn were negated by the guanylyl cyclase inhibitor, ODQ; 10 nmol/L sildenafil inhibited p47phox expression induced by U46619. CONCLUSIONS: Sildenafil is a potent inhibitor of superoxide formation in CVSMCs. This effect is mediated through the inhibition of PDE-5 which in turn augments the inhibitory action of the NO-cGMP axis on NAD[P]H oxidase expression and activity. This mechanism constitutes a new pharmacological action of sildenafil, consolidates the potential role of superoxide in ED, and indicates that thromboxane A(2) may be an important mediator of intrapenile oxidative stress.     

Sildenafil inhibits the up-regulation of phosphodiesterase type 5 elicited with nicotine and tumour necrosis factor-alpha in cavernosal vascular smooth muscle cells: mediation by superoxide

OBJECTIVES: To determine whether there is an association between vascular phosphodiesterase type 5 (PDE-5) and NADPH oxidase (NOX) in cavernosal vascular smooth muscle cells (CVSMCs), and to study the actions of the PDE-5 inhibitor sildenafil; the pro-erectile actions of nitric oxide (NO) are reduced by PDE-5 which hydrolyses cGMP to inactive GMP, thus an up-regulation of PDE-5 and over-production of O(2)(-) derived from NOX might promote erectile dysfunction (ED). MATERIALS AND METHODS: To study the effects of nicotine and tumour necrosis factor-alpha (TNF-alpha) on superoxide (O(2)(-)) production and PDE-5 expression, CVSMCs from rabbit penis were incubated with nicotine or TNF-alpha, and superoxide dismutase (SOD), catalase, sildenafil citrate, or apocynin (NADPH inhibitor) for 16 h. The expression of PDE-5 and of glyceraldehyde-3-phosphate dehydrogenase (internal standard) was assessed using Western blotting. O(2)(-) was measured spectrophotometrically. RESULTS: After a 16-h incubation, both nicotine (maximal at 10 microm) and TNF-alpha (10 ng/mL) significantly increased O(2)(-) formation in CVSMCs; this effect was blocked by co-incubating with SOD, catalase, and sildenafil (1 microm). Apocynin also inhibited O(2)(-) formation when added after 16-h incubation with nicotine (10 microm) or TNF-alpha. PDE-5 expression was also significantly increased in CVSMCs incubated with nicotine and TNF-alpha. This effect was negated by 16-h co-incubation with SOD, catalase, apocynin, and sildenafil. CONCLUSIONS: Nicotine and TNF-alpha up-regulate PDE-5 expression in CVSMCs through an a priori up-regulation of NOX and formation of O(2)(-). As PDE-5 hydrolyses cGMP, this effect might 'blunt' the pro-erectile actions of NO. Sildenafil inhibits O(2)(-) formation, and 'normalizes' PDE-5 expression. This represents a novel pathogenic mechanism underlying ED, and a novel mechanism of action of sildenafil.     

COMPUTER‐AIDED ULTRASONOGRAPHY (HISTOSCANNING): A NOVEL TECHNOLOGY FOR LOCATING AND CHARACTERIZING PROSTATE CANCER

OBJECTIVE

To test the hypothesis that exposure of a renal epithelial cell line, NRK52E, to calcium oxalate monohydrate crystals (COM) would up‐regulate NADPH oxidase subunit p47^phox, enhance superoxide production and increase monocyte chemoattractant protein‐1 (MCP‐1) and osteopontin mRNA levels.

MATERIALS AND METHODS

Confluent cultures of NRK52E cells were exposed to COM (66.7 µg/cm²) with or with no pretreatment with diphenileneiodium chloride (DPI, 10 × 10^?6m ) an inhibitor for NADPH oxidase, under serum‐free conditions. The conditioned medium was collected and total cellular RNA isolated from the cells, and subjected to enzyme‐linked immunosorbent assay and real‐time polymerase chain reaction (PCR). Production of reactive oxygen species (ROS) was estimated by dihydroethidium (DHE) staining using a fluorescence microscope. Immunohistochemistry and real‐time PCR were used to analyse p47^phox in NRK52E cells.

RESULTS

In COM treated NRK52E cells there was enhanced expression of p47^phox and production of superoxide. COM‐induced production of MCP‐1 and osteopontin was significantly reduced after treatment with DPI.

CONCLUSIONS

While the generation of a lot of ROS might play a major role in tissue injury or death, the regulated generation of low concentration of ROS, possibly by NADPH oxidase, may represent a second messenger system for generation of COM‐induced MCP‐1 and osteopontin production in the renal tubules.     

Investigation of the long-term patency of a transmural heparinized polycaprolactone and poly(D,L-lactic/glycolic acid) scaffold

Background

The transmural biodegradable polycaprolactone/poly(D,L-lactic/glycolic acid) (PCL/PLGA) scaffold is a promising modality for diffuse coronary atherosclerosis cases that are not suitable for bypass grafting. The purpose of this study was to evaluate the long-term performance of the PCL/PLGA scaffold in vivo in the setting of polymer and heparin degradation.

Materials and methods

After mechanical drilling through the ventricular wall was performed in the whole ventricular wall, two scaffolds were implanted into the ventricular wall. Animals were grouped into the single drilling group (SD group), the blank scaffold group (BS group), and the heparinized scaffold group (HS group) and were allowed to survived for 6 mo. Next, the patency and integrity of the scaffolds were evaluated by echocardiography and 3D-DOCTOR software. Endothelium coverage of the lumen was evaluated by scanning electron microscopy. Neovessels and collagen fiber within the scaffolds were identified by histologic staining. Metabolite production of prostacyclin (PGI₂) and thromboxane A₂ (TXA₂) in the plasma was measured by an enzyme-linked immunosorbent assay. The expression levels of PGI₂ synthase and cyclooxygenase 2 (COX-2) involved in PGI₂ production and COX-1 involved in TXA₂ production were measured by Western blot analysis.

Results

The heparinized scaffolds were patent for up to 6 mo and the lumen was covered with confluent endothelial cells. Histologic staining revealed collagen fiber remodeling and reconstruction of the neovascular network immediately surrounding the lumen. The expression of PGI₂ synthase and COX-2 in the HS group was significantly higher compared with the SD and BS groups (P < 0.01). The expression of COX-1 was similar in the three groups (P > 0.05). Consistent with synthetase expression, a PGI₂ metabolite (6-keto-PGF1a) also showed a significant increase in the HS group relative to the SD and BS groups (P = 0.021 and P = 0.015, respectively). Concomitantly, as a PGI₂ antagonist, the TXA₂ metabolite (TXB₂) did not exhibit a significant difference among the three groups (P = 0.17).

Conclusions

Despite polymer and heparin degradation, the scaffold could continuously maintain the structural integrity and lumen patency for up to 6 mo by reinforcement of host collagen fiber and the balance of PGI₂/TXA₂.     

An open-label, multicentre, randomized, crossover study comparing sildenafil citrate and tadalafil for treating erectile dysfunction in men naïve to phosphodiesterase 5 inhibitor therapy

Associate Editor Michael G. Wylie Editorial Board Ian Eardley, UK Jean Fourcroy, USA Sidney Glina, Brazil Julia Heiman, USA Chris McMahon, Australia Bob Millar, UK Alvaro Morales, Canada Michael Perelman, USA Marcel Waldinger, Netherlands

OBJECTIVES

To compare treatment preference, efficacy, and tolerability of sildenafil citrate (sildenafil) and tadalafil for treating erectile dysfunction (ED) in men naïve to phosphodiesterase 5 (PDE5) inhibitor therapy.

PATIENTS AND METHODS

This was an open‐label, crossover study of sildenafil and tadalafil (taken as needed). After a 4‐week baseline assessment, 367 men with ED (mean age 54 years) were randomized to receive sildenafil for 12 weeks followed by tadalafil for 12 weeks or vice versa (8‐week dose optimization and 4‐week assessment phases). During dose optimization, patients started taking 25‐ or 50‐mg sildenafil or 10‐mg tadalafil and could titrate to find their optimum dose (25‐, 50‐ or 100‐mg sildenafil; 10‐ or 20‐mg tadalafil). After completing both 12‐week periods, patients chose which treatment to continue during an 8‐week extension. Efficacy was measured with the International Index of Erectile Function (IIEF) and Sexual Encounter Profile (SEP) diary.

RESULTS

Of the 291 men who completed both treatments, 85 (29%) chose sildenafil and 206 (71%) chose tadalafil (P < 0.001) for the 8‐week extension. The IIEF erectile function domain scores were 14.2 at baseline, 23.9 at endpoint on sildenafil, and 24.3 at endpoint on tadalafil (P = 0.08, sildenafil vs tadalafil). The mean per patient percentage success scores for SEP2 (penetration) were: baseline (46%), sildenafil (post‐baseline 82%) and tadalafil (post‐baseline 85%; P = 0.06, sildenafil vs tadalafil), and for SEP3 (successful intercourse) were: baseline (19%), sildenafil (post‐baseline 72%), and tadalafil (post‐baseline 77%; P = 0.003, sildenafil vs tadalafil). The only treatment‐emergent adverse events that were reported by >5% of men were headache and flushing.

CONCLUSIONS

In men with ED who were naïve to PDE5 inhibitor therapy, sildenafil and tadalafil were both effective and well tolerated. After treatment with sildenafil and tadalafil, 29% of men chose sildenafil and 71% chose tadalafil for ED therapy during an 8‐week extension.

     

The effects of phosphodiesterase type 5 inhibitors on penile rigidity variables during a period with no sexual stimulation: a laboratory setting double‐blind study

Study Type – Therapy (RCT)
Level of Evidence 1b What’s known on the subject? and What does the study add? There is a positive effect of PDE5 inhibitors on several aspects of the men’s sex lives, chiefly erectile function, personal self‐esteem, and satisfaction from their sex lives. To our knowledge, our study is the first study to evaluate the effects of PDE5 inhibitors on erectile variables simultaneously in a laboratory setting. In the present study, significant penile rigidities were obtained with PDE5 inhibitors in a short period, with no sexual stimulation, in laboratory conditions. Our findings might support the use of PDE5 inhibitors in the men who need penile rehabilitation.

OBJECTIVE

To investigate the effects of phosphodiesterase type 5 (PDE5) inhibitors on erectile variables during a period with no sexual stimulation in a laboratory setting double‐blind study.

PATIENTS AND METHODS

In all, 80 men without erectile dysfunction (ED) but with lifelong premature ejaculation (PE) were included in the study. The men were divided equally in to four groups and received either placebo, vardenafil (10 mg), sildenafil (50 mg) or tadalafil (20 mg) in a double‐blind study design. The men attended the laboratory following 3 days of sexual abstinence and placebo or one of the PDE5 inhibitors was ingested after ≥2 h of fasting and non‐smoking. The men were then immediately placed in a silent room and real‐time penile rigidity and tumescence monitoring with Rigiscan Plus (Rigiscan Plus® System, Osbon Medical Systems, Augusta, GA, USA) began. The men read some magazines or newspapers that contained no sexually stimulating material for 1.5 h. There was no interaction between the men and observer during the test period. Times to first measured and total durations of base and tip rigidities, and also total and per minute rigidity were evaluated.

RESULTS

The recorded base and/or tip rigidity ratios were 40% (eight of 20), 71% (12/17), 47% (nine of 19) and 70% (14/20) in men who took placebo, sildenafil, tadalafil and vardenafil, respectively (P= 0.126). The ratio of men who could obtain ≥60% base and/or tip rigidities were 10% (two of 20), 41% (seven of 17), 26% (five of 19) and 55% (11/20) in placebo, sildenafil, tadalafil and vardenafil groups, respectively (P < 0.05). The median time to first measured base rigidity was 58.0, 21.5, 54.5 and 57 min with placebo, sildenafil, tadalafil and vardenafil, respectively (P= 0032). The median total duration of recorded base rigidity was 4.0, 27.5, 10.0 and 11.5 min in men who took placebo, sildenafil, tadalafil and vardenafil, respectively (P= 0.013). The median total base rigidity (area under the curve) was 72.8, 699.0, 360.5 and 553.0 with placebo, sildenafil, tadalafil and vardenafil, respectively (P= 0.016).

CONCLUSIONS

Significant penile rigidities were obtained with PDE5 inhibitors during the short test period, with no sexual stimulation, in laboratory conditions. This finding might support the use of PDE5 inhibitors in men who need penile rehabilitation.     

Penile rehabilitation protocol after robot‐assisted radical prostatectomy: assessment of compliance with phosphodiesterase type 5 inhibitor therapy and effect on early potency

Study Type – Therapy (case series)
Level of Evidence 4

OBJECTIVE

To evaluate factors that affect compliance in men who enrol in a phosphodiesterase type 5 inhibitor (PDE5I) protocol after nerve‐sparing robot‐assisted prostatectomy (RAP), and report on short‐term outcomes, as PDE5Is may help restore erectile function after RAP and patient adherence to the regimen is a factor that potentially can affect outcome.

PATIENT AND METHODS

We prospectively followed 77 men who had nerve‐sparing RAP and enrolled in a postoperative penile rehabilitation protocol. The men received either sildenafil citrate or tadalafil three times weekly. The minimum follow‐up was 8 weeks. Potency was defined as erection adequate for penetration and complete intercourse. Compliance was defined as men adhering to the regimen for ≥2 months.

RESULTS

The mean age of the cohort was 57.8 years and the median follow‐up was 8 months. In all, 32% of the men discontinued the therapy <2 months after RAP and were deemed noncompliant with an additional 39% discontinuing therapy by 6 months, with the high cost of medication being the primary reason (65%). Long‐term compliance and preoperative erectile dysfunction were independent predictors of potency return after adjusting for age and nerve sparing.

CONCLUSIONS

The high cost of medication remains a significant barrier to maintaining therapy. Noncompliance to PDE5I therapy in a tertiary care centre was much higher than reported in clinical trial settings. With longer‐term follow‐up, we need to further define the factors that improve overall recovery of sexual function after RAP.     

Early intervention with phosphodiesterase‐5 inhibitors after prostate brachytherapy improves subsequent erectile function

Study Type – Therapy (case series)
Level of Evidence 4

OBJECTIVE

To examine the early use of phosphodiesterase‐5 inhibitor (PDE‐5i; sildenafil citrate) in preventing subsequent erectile dysfunction (ED) after (monotherapy) prostate brachytherapy (PB, an accepted option for Gleason 6 or low‐volume Gleason 7 prostate cancer), as PB is currently being offered more frequently in younger patients, and ED can be a side‐effect often within the first 12 months after treatment.

PATIENTS AND METHODS

We examined a single‐surgeon series of 69 patients who had been treated with PB from 2002 to 2005. All patients had a follow‐up of ≥1 year; prospectively, and patients had baseline, 6‐ and 12‐month assessments using the Sexual Health Inventory for Men (SHIM) and International Index of Erectile Function (IIEF)‐6 scores. The 69 patients were divided into early treatment with PDE‐5i (31) and not treated with PDE‐5i (38), and their SHIM and IIEF‐6 scores were compared at baseline, 6 and 12 months. Daily sildenafil (25–50 mg) was given immediately after PB for 12 months. Overall, for the entire group, the mean prostate‐specific antigen (PSA) level was 6.8 ng/mL; 78% had Gleason 6 cancer and 20% had Gleason 7 (3 + 4) cancer. The mean age in the early PDE‐5i group was 64.8 years, and was 66.0 years in the no‐PDE‐5i group. The mean radiation dose in the early PDE‐5i group was 50.2 Gy, and 43.9 Gy in the other group (P= 0.08).

RESULTS

In the no‐PDE‐5i group, the mean baseline SHIM score of 17.1 decreased rapidly to 9.1 at 6 months (P= 0.01) and stayed at 9.3 at 12 months (P= 0.01). In the early PDE‐5i group, the mean baseline SHIM score of 21.8 decreased slightly to 17.6 at 6 months (P= 0.2), and was maintained at 17.9 at 12 months (P= 0.2). Using the Wilcoxon rank‐sum test, the 6‐ and 12‐month SHIM scores in the two groups (P < 0.001). The IIEF‐6 questionnaire confirmed the SHIM analysis.

CONCLUSIONS

After PB patients had a significant decline in SHIM/IIEF‐6 scores at 6 and 12 months. Our results indicate a 50% decrease in the quality of their erections. This provides an opportunity to initiate early intervention with PDE‐5i or perhaps vacuum constriction devices or intraurethral alprostadil. In this study, the early use of PDE‐5i after PB maintained erectile function at both 6 and 12 months.     

A G‐protein Subunit‐α11 Loss‐of‐Function Mutation,Thr54Met,Causes Familial Hypocalciuric Hypercalcemia Type 2 (FHH2)

Familial hypocalciuric hypercalcemia (FHH) is a genetically heterogeneous disorder with three variants, FHH1 to FHH3. FHH1 is caused by loss‐of‐function mutations of the calcium‐sensing receptor (CaSR), a G‐protein coupled receptor that predominantly signals via G‐protein subunit alpha‐11 (Gα₁₁) to regulate calcium homeostasis. FHH2 is the result of loss‐of‐function mutations in Gα₁₁, encoded by GNA11, and to date only two FHH2‐associated Gα₁₁ missense mutations (Leu135Gln and Ile200del) have been reported. FHH3 is the result of loss‐of‐function mutations of the adaptor protein‐2 σ‐subunit (AP2σ), which plays a pivotal role in clathrin‐mediated endocytosis. We describe a 65‐year‐old woman who had hypercalcemia with normal circulating parathyroid hormone concentrations and hypocalciuria, features consistent with FHH, but she did not have CaSR and AP2σ mutations. Mutational analysis of the GNA11 gene was therefore undertaken, using leucocyte DNA, and this identified a novel heterozygous GNA11 mutation (c.161C>T; p.Thr54Met). The effect of the Gα₁₁ variant was assessed by homology modeling of the related Gα_q protein and by measuring the CaSR‐mediated intracellular calcium (Ca²⁺_i) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca²⁺_o) using flow cytometry. Three‐dimensional modeling revealed the Thr54Met mutation to be located at the interface between the Gα₁₁ helical and GTPase domains, and to likely impair GDP binding and interdomain interactions. Expression of wild‐type and the mutant Gα₁₁ in HEK293 cells stably expressing CaSR demonstrate that the Ca²⁺_i responses after stimulation with Ca²⁺_o of the mutant Met54 Gα₁₁ led to a rightward shift of the concentration‐response curve with a significantly (p < 0.01) increased mean half‐maximal concentration (EC₅₀) value of 3.88 mM (95% confidence interval [CI] 3.76–4.01 mM), when compared with the wild‐type EC₅₀ of 2.94 mM (95% CI 2.81–3.07 mM) consistent with a loss‐of‐function. Thus, our studies have identified a third Gα₁₁ mutation (Thr54Met) causing FHH2 and reveal a critical role for the Gα₁₁ interdomain interface in CaSR signaling and Ca²⁺_o homeostasis. © 2016 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).     

Comparative effects of T‐type and L‐type Ca2+‐antagonists against noradrenaline‐induced contractions of human vas deferens

OBJECTIVE

To investigate the effects of the relatively selective T‐type Ca²⁺‐antagonists, mibefradil and flunarizine, and the L‐type Ca²⁺‐antagonist, nifedipine, on the contractions of longitudinal and circular muscles of human vas deferens, to elucidate the possible involvement of T‐type voltage‐gated Ca²⁺ channels (VOCs) in the contractile function of the tissue.

MATERIALS AND METHODS

Human vas deferens specimens from elective vasectomies were cut into strips of longitudinal muscle or transversely into rings of circular muscle. These were set up for tension recording and superfused with Krebs’ medium (36° C). Contractions were evoked by noradrenaline or high [K⁺]_o (in the presence of the L‐type Ca²⁺ agonist, FPL 64176) and the effects of Ca²⁺ antagonists were determined.

RESULTS

Noradrenaline (0.1–100 µmol/L) evoked rhythmic and tonic contractions of longitudinal and circular muscles, which were potently inhibited by nifedipine (≤0.1 µmol/L). Mibefradil (1–10 µmol/L) inhibited the contractions but was comparatively more effective in longitudinal than circular muscle. Flunarizine was ineffective except against contractions to low concentrations of noradrenaline. The drugs’ potencies as antagonists of L‐type VOCs were determined against contractions to high K⁺ (120 mmol/L in the presence of FPL 64176, 1 µmol/L). The contractions in longitudinal and circular muscle had different times to peak and decline but were inhibited comparably by nifedipine (50% inhibitory concentration, IC₅₀, longitudinal and circular muscle, ≈2 nmol/L) or by mibefradil (IC₅₀ longitudinal muscle, 1.1 µmol/L; circular muscle, 2.4 µmol/L) and were less sensitive to flunarizine (up to 30 µmol/L).

CONCLUSION

These results indicate that noradrenaline‐induced contractions of human vas deferens depend primarily on nifedipine‐sensitive L‐type VOCs, as opposed to mibefradil/flunarizine‐sensitive T‐type VOCs. The effects of mibefradil and flunarizine, at concentrations found to be effective against noradrenaline‐induced contractions, involve the blockade of L‐type VOCs. The modest differential effect of mibefradil in longitudinal and circular muscle is discussed in relation to factors that modulate activation and drug‐sensitivity of L‐type VOCs.     

Sildenafil Effect on the Human Bladder Involves the L-cysteine/Hydrogen Sulfide Pathway: A Novel Mechanism of Action of Phosphodiesterase Type 5 Inhibitors

Background

Phosphodiesterase type 5 inhibitors (PDE5-Is) are effective in the treatment of lower urinary tract symptom (LUTS), although their mechanism of action is still unclear. PDE5-Is cause bladder detrusor relaxation, and this effect is partially independent of nitric oxide. Hydrogen sulfide (H₂S) is a newly discovered transmitter with myorelaxant properties. It is predominantly formed from L-cysteine by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE).

Objective

To evaluate whether the L-cysteine/H₂S pathway contributes to the relaxing effect of sildenafil on the human detrusor dome.

Design, setting, and participants

Samples of bladders obtained from men undergoing open prostatectomy for benign prostatic hyperplasia (BPH) were used. The presence of CBS and CSE enzymes was assessed by western blot. H₂S production was measured by a colorimetric assay in basal and stimulated conditions with L-cysteine and in response to sildenafil (1, 3, 10, and 30 μM), 8-bromo–cyclic guanosine monophosphate (8-bromo–cGMP; 100 μM) or dibutyryl–cyclic adenosine monophosphate (dibutyryl-cAMP; 100 μM). A curve concentration effect of sodium hydrosulfide (NaHS), H₂S donor (0.1 μM to 10 mM), L-cysteine (0.1 μM to 10 mM), and sildenafil (0.1–10 μM) was performed on precontracted detrusor dome strips. To investigate H₂S signaling in a sildenafil effect, CBS and CSE inhibitors were used.

Outcome measurements and statistical analysis

Analysis of variance was used, followed by the Bonferroni post hoc test.

Results and limitations

CBS and CSE are present in the human bladder dome and efficiently convert L-cysteine into H₂S. Both NaHS and L-cysteine relaxed human strips. Sildenafil caused (1) a relaxation of bladder dome strips and (2) a concentration-dependent increase in H₂S production. Both effects were significantly reduced by CBS and CSE inhibitors. Similar to sildenafil, both 8-bromo-cGMP and dibutyryl-cAMP caused an increase in H₂S production.

Conclusions

The sildenafil relaxant effect on the human bladder involves the H₂S signaling pathway. This effect may account in part for the efficacy of PDE5-Is in LUTS. A better definition of the pathophysiologic role of the H₂S pathway in the human bladder may open new therapeutic approaches.     

Differences in side‐effect duration and related bother levels between phosphodiesterase type 5 inhibitors

OBJECTIVE

To assess whether the longer half‐life of tadalafil is associated with longer lasting or more severe side‐effects than the other phosphodiesterase type 5 inhibitors (PDE‐5Is), as clinical trials have shown that the efficacy and safety of the three available are similar, but tadalafil has a half‐life four times longer than the other two drugs.

PATIENTS AND METHODS

Treatment‐naive men beginning PDE5‐I therapy were recruited from a specialist clinic. Data on the type and duration of drug‐associated side‐effects were collected prospectively. Levels of bother were assessed with a visual analogue scale (VAS). Differences in type, duration and bother of side‐effect were compared between drugs.

RESULTS

In all, 409 men provided data; there were no differences between drugs in the proportion of men responding, or the overall prevalence of side‐effects. The mean duration of side‐effects with tadalafil was 14.9 h, compared to 3.9 and 7.7 h for sildenafil and vardenafil. Of men taking tadalafil, 30% had side‐effects lasting >12 h. There were no differences in mean VAS scores between the drugs. Individual side‐effects caused similar levels of bother, except for facial flushing, which was less bothersome.

CONCLUSIONS

Men taking tadalafil are at risk of prolonged side‐effects, although levels of bother associated with these side‐effects are not significantly greater than those seen with short‐acting PDE5‐Is.     

Development of Arterial Calcification in Adiponectin‐Deficient Mice: Adiponectin Regulates Arterial Calcification

Arterial calcification is common, but the mechanisms remain unclear. This study was undertaken to investigate the arterial calcification in adiponectin‐deficient mice in vivo and the effects of adiponectin on cultured vascular smooth muscle cells in vitro. Alizarin red S staining was used to detect arterial calcification of adiponectin^?/? mice. Alkaline phosphatase activity, osteocalcin secretion, and Runx2 protein expression were examined in cultured calcifying vascular smooth muscle cells (CVSMCs). The involved signal pathway was studied using a mitogen‐activated protein kinase (MAPK) inhibitor and adiponectin receptor 1 (AdipoR1) siRNA. Adiponectin^?/? mice developed slight arterial calcification after being fed with normal chow diet for 30 wk. Adenovirus‐mediated supplement of adiponectin attenuated arterial calcification in these mice. On cultured CVSMCs, adiponectin inhibited ALP activity, osteocalcin secretion, Runx2 protein expression, and the formation of mineralized nodules. Adiponectin receptor 1 (AdipoR1) protein was detected in CVSMCs, and adiponectin activated p38 mitogen‐activated protein kinase. Furthermore, inhibition of AdipoR1 expression or p38 activation reversed the effects of adiponectin on ALP activity. These results showed that adiponectin inhibited osteoblastic differentiation of CVSMCs through the AdipoR1/p38 signaling pathway. Our findings showed that adiponectin^?/? mice developed arterial calcification, and this could be attributed to the loss of inhibitory action of adiponectin on osteoblastic differentiation of CVSMCs. It suggested that adiponectin plays a protective role against arterial calcification.     

Safety and efficacy of sildenafil citrate in treating erectile dysfunction in patients with combat‐related post‐traumatic stress disorder: a double‐blind,randomized and placebo‐controlled study

OBJECTIVE

To evaluate the safety and efficacy of sildenafil citrate for treating erectile dysfunction (ED) in patients with combat‐related post‐traumatic stress disorder (PTSD).

PATIENTS AND METHODS

In all, 266 combat‐exposed war veterans with ED (aged 37–59 years) were recruited. They met the Diagnostic and Statistical Manual of Mental Disorders‐IV criteria for PTSD according to the Structured Clinical Interview for Patients, Investigator Version. The patients were also evaluated with the Clinician‐Administered PTSD Scale, both to establish the diagnosis of PTSD and to measure symptom severity. Only patients with psychogenic ED were included in the study. Patients with comorbid conditions (diabetes mellitus, hypercholesterolaemia, hypertension, Peyronie’s disease) and smokers of more than five cigarettes daily were excluded. The patients were randomly divided into a group of 133 who received 100 mg of on‐demand sildenafil 0.75–2 h before sexual stimulation, and 133 who received placebo. Patients were asked to use ≥16 doses or attempts at home. The efficacy of the treatments was assessed every four attempts during treatment, and at the end of the study, using responses to the 15‐question International Index of Erectile Function (IIEF), Sexual Encounter Profile diary questions 2 and 3, Erectile Dysfunction Inventory of Treatment Satisfaction questionnaire, patients’ event logs of sexual activity, and a Global Assessment Question about erections.

RESULTS

Sildenafil did not produce significantly and substantially greater improvement than placebo in each of the primary and secondary outcome measures (P = 0.08). A normal EF domain score (≥26) at endpoint was reported by 13 (9.8%), and 11 (8.3%) of patients on the sildenafil and placebo regimens, respectively (P = 0.09). Patients treated with sildenafil had no statistically significantly greater improvement in the five sexual function domains of the IIEF questionnaire than those treated with placebo (P = 0.08). The incidences of treatment‐emergent adverse events were significantly greater in the sildenafil arm than in the placebo group (P = 0.01).

CONCLUSIONS

Sildenafil is no better than placebo in treating PTSD‐emergent ED. Further randomized clinical trials are warranted in combat veterans and other populations with PTSD to better elucidate the role of phosphodiesterase type 5 inhibitors in treating PTSD‐emergent ED.     

Effects of 25‐hydroxyvitamin D3 on proliferation and osteoblast differentiation of human marrow stromal cells require CYP27B1/1α‐hydroxylase

1,25‐Dihydroxyvitamin D₃ [1,25(OH)₂D₃] has many noncalcemic actions that rest on inhibition of proliferation and promotion of differentiation in malignant and normal cell types. 1,25(OH)₂D₃ stimulates osteoblast differentiation of human marrow stromal cells (hMSCs), but little is known about the effects of 25‐hydroxyvitamin D₃ [25(OH)D₃] on these cells. Recent evidence shows that hMSCs participate in vitamin D metabolism and can activate 25(OH)D₃ by CYP27B1/1α‐hydroxylase. These studies test the hypothesis that antiproliferative and prodifferentiation effects of 25(OH)D₃ in hMSCs depend on CYP27B1. We studied hMSCs that constitutively express high (hMSCs^hi‐1α) or low (hMSCs^lo‐1α) levels of CYP27B1 with equivalent expression of CYP24A1 and vitamin D receptor. In hMSCs^hi‐1α, 25(OH)D₃ reduced proliferation, downregulated proliferating cell nuclear antigen (PCNA), upregulated p21^Waf1/Cip1, and decreased cyclin D1. Unlike 1,25(OH)₂D₃, the antiapoptotic effects of 25(OH)D₃ on Bax and Bcl‐2 were blocked by the P450 inhibitor ketoconazole. The antiproliferative effects of 25(OH)D₃ in hMSCs^hi‐1α and of 1,25(OH)₂D₃ in both samples of hMSCs were explained by cell cycle arrest, not by increased apoptosis. Stimulation of osteoblast differentiation in hMSCs^hi‐1α by 25(OH)D₃ was prevented by ketoconazole and upon transfection with CYP27B1 siRNA. These data indicate that CYP27B1 is required for 25(OH)D₃'s action in hMSCs. Three lines of evidence indicate that CYP27B1 is required for the antiproliferative and prodifferentiation effects of 25(OH)D₃ on hMSCs: Those effects were not seen (1) in hMSCs with low constitutive expression of CYP27B1, (2) in hMSCs treated with ketoconazole, and (3) in hMSCs in which CYP27B1 expression was silenced. Osteoblast differentiation and skeletal homeostasis may be regulated by autocrine/paracrine actions of 25(OH)D₃ in hMSCs. © 2011 American Society for Bone and Mineral Research.     

The effects of three phosphodiesterase type 5 inhibitors on ejaculation latency time in lifelong premature ejaculators: a double-blind laboratory setting study

Study Type – Therapy (RCT)
Level of Evidence 1b What’s known on the subject? and What does the study add? Several authors have reported their experience with PDE5 inhibitors alone or in combination with selective serotonin re‐uptake inhibitors for treating premature ejaculation. However, to our knowledge, this is the first laboratory design study to evaluate the effects of three PDE5 inhibitors throughout the ejaculation process in men with lifelong premature ejaculation. In this laboratory setting study PDE5 inhibitors seem to prolong ELT but the difference from placebo is significant only in vardenafil. The quality of penile rigidity is better with PDE5 inhibitors in the post‐ejaculatory period but the difference is significant only in sildenafil and vardenafil.

OBJECTIVE

? To evaluate the effects of three phosphodiesterase type 5 (PDE5) inhibitors on the ejaculation process in men with lifelong premature ejaculation using a double‐blind laboratory setting.

PATIENTS AND METHODS

? Eighty men with lifelong premature ejaculation, 20 in each group, received placebo, vardenafil (10 mg), sildenafil (50 mg) or tadalafil (20 mg) in a double‐blind study design. Placebo or PDE5 inhibitor was ingested after at least 2 h fasting and non‐smoking. The subjects were placed in a silent room immediately and real‐time penile rigidity and tumescence was monitored. ? Subjects read some magazines or newspapers without any sexually stimulating material for 1.5 h. At the end of this period audiovisual sexual stimulation began with a video film and after the 8th minute the subject began vibratory stimulation to the frenular area. ? At the beginning of ejaculation the patient stopped stimulation. When the patient began and stopped stimulation, the light near the observer turned on and off and the observer calculated the ejaculation period with a chronometer. The elapsed time was the ejaculation latency time (ELT) in seconds. ? There was no interaction between subjects and observer during the test. The ELT, and the qualities of base and tip rigidities during ELT and after ejaculation were calculated.

RESULTS

? Median age of patients was 29 (range 22–39) years and median duration of premature ejaculation was 60 (range 7–180) months and there was no significant difference between groups. Median duration of vibratory stimulation (ELT) of subjects who received placebo was 48.5 s: 53.5 s for sildenafil, 70.0 s for tadalafil and 82.5 s for vardenafil. Compared with the placebo group, ELT was significantly longer only in subjects receiving vardenafil (P = 0.019). ? In the post‐ejaculatory refractory period, times to last recorded base rigidities were significantly longer than placebo in vardenafil and sildenafil groups with better erection quality (P < 0.01 for each).

CONCLUSIONS

? The PDE5 inhibitors seem to prolong ELT and the quality of penile rigidity is better with PDE5 inhibitors in post‐ejaculatory period. ? These findings suggest that PDE5 inhibitors might have some beneficial effects in men with lifelong premature ejaculation.     

Inhibition of sympathetic neuroeffector transmission in human corpus cavernosum

Study Type – Aetiology (case control) Level of Evidence 2b What's known on the subject? and What does the study add? In the present study the mechanisms regulating EFS‐evoked neurogenic contraction in the human corpus cavernosum (HCC) were investigated. Overall, our data adds to current knowledge that the NO‐independent heme dependent activation of sGC and the RhoA/Rho‐kinase signaling pathways play an important role in the regulation of neurogenic contractile activity in HCC tissue.

OBJECTIVE

• ? To investigate the mechanisms of adrenergically mediated smooth muscle contraction in the human corpus cavernosum (HCC) using an organ bath approach.

METHODS

• ? Human corpus cavernosum specimens were obtained from patients (aged 59–72 years) with erectile dysfunction (ED), undergoing penile prosthesis implantation surgery.
• ? Isolated HCC strips (1 × 1 × 6 mm) were suspended in tissue bath chambers for isometric tension recording.
• ? The effects of various drugs on neurogenic contractions evoked by electrical field stimulation (EFS) were investigated. The drugs included nitric oxide (NO) donors, phosphodiesterase 5 (PDE5) inhibitor, Rho kinase (ROCK) inhibitor, NO‐independent stimulator, L‐type Ca2+ channel blocker and α‐receptor antagonist.

RESULTS

• ? Pre‐incubation with the NO donor sodium nitroprusside (SNP; 10⁴ M) significantly reduced the initial peak increase in tension evoked by EFS (by 71%, P < 0.05). The PDE5 inhibitor sildenafil (10^?4 M) reduced the increase in tension by 69%, while a combination of sildenafil and ROCK inhibitor, fasudil, inhibited tension by 81%.
• ? The EFS‐induced contractile response at 80 Hz was decreased by 65% with fasudil and by 70% with isradipine (P < 0.001), while a combination of these drugs decreased the response by 88%. An NO‐independent stimulator soluble guanylate cyclase (sGC), BAY 41‐8543, significantly reduced the response (by 82%, P < 0.001) Phentolamine, an α‐receptor antagonist, nearly eliminated the contractile response (98%, P < 0.001).

CONCLUSIONS

• ? These data suggest that neurogenic contractions are mediated by an increase in Ca(2+) influx via L‐type voltage‐gated Ca(2+) channels and that an increase in Ca(2+) sensitivity is mediated by the ROCK pathway and the PDE5 enzyme system as well as by the inhibitory NO/sGC/cGMP pathway.
• ? The neurogenic contractile response in HCC is mediated by several intracellular pathways, including adrenergic receptors, Ca(2+) entry, Ca(2+) sensitization and activation of the PDE5 enzyme. The Rho‐kinase (ROCK) inhibitor fasudil, L‐type Ca(2+) channel antagonist isradipine, and PDE5 inhibitor sildenafil, as well as a NO‐independent stimulator of sGC, had similar inhibitory effects, suggesting parallel mechanisms in the HCC.