Phosphodiesterase Type 5 Expression Research Articles

Combined levothyroxine and testosterone treatment for restoring erectile dysfunction in propylthiouracil-induced hypothyroid rats.

Sexual dysfunction may indicate severe endocrine diseases. Recent research has suggested a link between hypothyroidism, low testosterone (T) levels, and erectile dysfunction (ED); however, the exact cause is unknown. We sought to investigate possible beneficial effects of levothyroxine and T alone or in combination on ED in propylthiouracil (PTU)-induced hypothyroid rats. Adult Wistar rats (n = 35) were divided into 5 groups: control, PTU-induced hypothyroidism, PTU + levothyroxine, PTU + Sustanon (a mixture of 4 types of T: propionate, phenylpropionate, isocaproate, and decanoate) and PTU + levothyroxine + Sustanon. PTU was given in drinking water for 6 weeks. Four weeks after PTU administration, levothyroxine (20μg microgram kg/day, oral) and Sustanon (10mg/kg/week, intramuscular) were given for 2weeks. Serum levels of total T, triiodothyronine (T3), and thyroxine (T4) were determined. In vivo erectile response and in vitro relaxant responses were measured. Localization of neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS), and phosphodiesterase type 5 (PDE5) were determined using immunohistochemical analysis. The relative area of smooth muscle to collagen was measured using Masson trichrome staining. Outcome variables included in vivo erectile function, in vitro relaxant and contractile responses of corpus cavernosum (CC) strips; protein localization of eNOS, nNOS, and PDE5; and smooth muscle content in penile tissue. The rat model of hypothyroidism showed a significant decline in serum levels of total T, T3, and T4. Levothyroxine increased T3 and T4 levels, whereas Sustanon normalized only total T levels. Combined treatment enhanced all hormone levels. Rats with hypothyroidism displayed the lowest erectile response (P < 0.001 vs controls). Combined treatment returned reduced responses, while partial amelioration was observed after levothyroxine and Sustanon treatment alone. Acetylcholine (P < 0.01 vs controls), electrical field stimulation (P < 0.001 vs controls), and sildenafil-induced relaxant responses (P < 0.05 vs controls) were decreased in the CC strips from hypothyroid rats. The combined treatment increased the reduction in relaxation responses. Levothyroxine and Sustanon restored decreases in eNOS and nNOS expression in the hypothyroid group. There was no significant difference in PDE5 expression among groups. Monotreatment partially enhanced reduced smooth muscle mass, while combined therapy completely recovered. The combination of thyroid hormones and T is likely to be a therapeutic approach for treatment of hypothyroidism-induced ED in men. Beneficial effects of levothyroxine and Sustanon treatment were shown in vitro and in vivo in PTU-induced hypothyroid rats. The main limitation of the study was the lack of measurement of androgen-sensitive organ weights and luteinizing hormone, follicle-stimulating hormone, and prolactin levels. These findings demonstrate that neurogenic and endothelium-dependent relaxation responses are reduced by hypothyroidism, which is detrimental to T levels and erectile responses. Levothyroxine and Sustanon combination medication was able to counteract this effect.

Abstract 10753: Phosphodiesterase Type 5 Inhibition and C-c Chemokine Receptor-2 Disruption Synergistically Protect Against Pulmonary Arterial Hypertension in Monocrotaline-Treated Rats: Potential Synergism of Anti-inflammatory Agents and Pulmonary Vasodilator Therapy

BACKGROUND: Whether conventional pulmonary vasodilator therapy and anti-inflammatory agents synergistically protect against pulmonary arterial hypertension (PAH) is unknown. Hypothesis: We tested the hypothesis that a phosphodiesterase type 5 (PDE5) inhibitor and disruption of C-C chemokine receptor (CCR)-2, a receptor for MCP-1, synergistically protect against PAH in monocrotaline (MCT)-treated rats. Methods: Study1:Seven-week-old male Ccr2 (-/-) rats, generated by using the CRISPAR/ Cas9 genome editing technology, or wild-type littermates, treated with MCT (60mg/kg) or vehicle, were subjected to hemodynamic, morphometric and immunohistochemical analyses and mRNA and protein expression studies on day21 (n=40, 4 groups). Study 2: Tadarafil (10mg/kg/d), a PDE5 inhibitor, or vehicle was administered in Ccr2 (-/-) or wild-type MCT rats on days 21-42 (n=71, 4 groups) for evaluating survival and were evaluated hemodynamically on day28. Results: Study1: MCT treatment increased right ventricular systolic pressure (RVSP), an index of right ventricular hypertrophy (Fulton index), indices of pulmonary vascular diseases (%medial wall thickness, %muscularization) and the number of perivascular CD68-positive macrophages in WT rats, all of which were decreased by Ccr2 (-/-) (p&lt;.05). Similarly, an increase in mRNA expression of inflammatory cytokines (IL6 ,TNFa, MCP1, IL1b, TGFb) and a decrease in BMPR2 pathways (BMPR2, pSmad1/5/9 and ID1) as well as an increase in endothelial apoptosis (% cleaved Caspase 3 positive CD31 cells) and a decrease in NO pathways (eNOS and phospho-eNOS/eNOS) in lungs in wild-type MCT rats were reversed by Ccr2 (-/-) on day21 (p&lt;.05) with comparable PDE5 expression. Study2: Tadarafil administration or CCR2(-/-)(p=.03 and p=.02) alone improved the survival in wild type MCT rats. A double intervention synergistically improved the survival in separately treated groups (p=.02 vs CCR2(-/-), p=.04 vs Tadarafil) in MCT rats. Tadarafil treatment on days21-28 decreased RVSP and Fulton index in CCR2(-/-) MCT rats (p&lt;.05). Conclusions PDE5 inhibition and CCR-2 disruption synergistically protect against PAH in MCT-treated rats, suggesting an adjunctive role of a CCR2 inhibitor on top of administering a PDE5 inhibitor for PAH.

Combination therapy of doxorubicin and Sildenafil inhibits the growth of pediatric rhabdomyosarcoma

BackgroundOverexpression of phosphodiesterase type 5 (PDE5) has been detected in many types of malignant tumors. Sildenafil, a potent and selective inhibitor of a cGMP-specific PDE5, has been found to enhance the cytotoxic activity of different chemotherapeutic agents including doxorubicin. The combined therapy of doxorubicin with Sildenafil might reduce the possible side effects of chemotherapy while maintaining synergistic anticancer activity. The present study explored for the first time the effects of Sildenafil, alone and in combination with doxorubicin, on pediatric RMS cells.MethodsHuman RMS cells lines RH30 and RD were used. Western blotting and RT-PCR were performed to analyze PDE5 expression in RMS cells. Cell viability was determined using MTT assay. Cell migration was analyzed via transwell chambers, clonal growth and mitotic cell death were analyzed using the clonogenic assay. FACS analysis was performed to evaluate reactive oxygen species (ROS) and apoptosis.ResultsSildenafil significantly decreased cell viability and migration of RD and RH30 cells. The exposure of RMS cells to doxorubicin resulted in a dose-dependent decrease in their viability. Simultaneous administration of Sildenafil enhanced this effect. The incubation of the RMS cells with Sildenafil in the presence of doxorubicin significantly increased the proportion of apoptotic cells and ROS production compared to the treatment with Sildenafil alone.ConclusionsThe results of our study indicate a link between PDE5 inhibition by Sildenafil and decreased calcium signalling leading to significantly impaired viability, migration, and colony forming of RMS cells. Increased ROS production and apoptosis are mechanisms relevantly contributing to this observation.

In vitro inhibition of phosphodiesterase type 4 enhances rat corpus cavernosum nerve-mediated relaxation induced by gasotransmitters

AimsNitric oxide (NO) and hydrogen sulfide (H2S) are involved in nerve-mediated corpus cavernosum (CC) relaxation. Expression of phosphodiesterase type 5 (PDE5) and type 4 (PDE4), cyclic guanosine monophosphate (cGMP)- and cyclic adenosine monophosphate (cAMP)-specific, respectively, has been described and PDE5- and PDE4-inhibitors induce cavernous smooth muscle relaxation. Whereas the NO/cGMP signaling pathway is well established in penile erection, the cAMP-mediated mechanism is not fully elucidated. The aim of this study is to investigate the localization and the functional significance of PDE4 in rat CC tone regulation. Main methodsWe performed immunohistochemistry for the detection of the PDE4A isoenzyme. Isometric tension recordings for roflumilast and tadalafil, PDE4 and PDE5 inhibitors, respectively, electrical field stimulation (EFS) and β-adrenoceptor agonist isoproterenol and endogenous H2S production measurement. Key findingsA marked PDE4A expression was detected mainly localized in the nerve cells of the cavernous smooth muscle. Furthermore, roflumilast and tadalafil exhibited strong corpus cavernous relaxations. Endogenous H2S production was decreased by NO and H2S synthase inhibitors and increased by roflumilast. Isoproterenol- and EFS-induced relaxations were increased by roflumilast. SignificanceThese results indicate that PDE4A is mainly expressed within the nerves cells of the rat CC, where roflumilast induces a potent corpus cavernous relaxation per se and potentiates the response induced by β-adrenoceptor activation. The fact that roflumilast enhances H2S production, as well as EFS-elicited responses suggests that PDE4 inhibitors modulate, in a positive feedback fashion, nerve-mediated relaxation induced by gasotransmitters, thus indicating a key role for neuronal PDE4 in penile erection.

Beneficial Effects of Human Umbilical Cord Blood Mononuclear Cells on Persistent Erectile Dysfunction After Treatment of 5-Alpha Reductase Inhibitor in Rats

Effects of human umbilical cord blood (HUCB) as a valuable source for stem cell-based therapies have not been studied in persistent post-5-alpha reductase inhibitors (5ARI) erectile dysfunction (PPED). To determine the effect of intracavernosal injection of HUCB mononuclear cells (MNCs) on ED associated with dutasteride treatment. Twenty five adult male Sprague-Dawley rats were divided into 5 groups (n=5 per group): (i) control, (ii) 8-week dutasteride (0.5 mg/kg/day, in drinking water), (iii) 12-week dutasteride, (iv) 8-week dutasteride+HUCB-MNCs (1×106) and (v) 12-week dutasteride+HUCB-MNCs. HUCB-MNCs were administered intracavernosally after eight weeks of dutasteride treatment. Experiments were performed at 4 weeks following the injection of HUCB-MNCs. Erectile responses and isometric tension of corpus cavernosum (CC) were measured. The protein expressions of phosphodiesterase type 5 (PDE5), endothelial nitric oxide synthase (eNOS), neuronal NOS (nNOS), hypoxia-inducible factor (HIF)-1α and smooth muscle/collagen contents in penile tissue were evaluated by Western blotting, immunohistochemistry, and Masson's trichrome staining, respectively. In vivo erectile function, in vitro relaxant and contractile responses of CC, protein expression and localization of PDE5, eNOS, nNOS, HIF-1α, and smooth muscle content in penile tissue. Erectile responses in the dutasteride-treated groups were significantly decreased compared with controls (P < .001), persisting after 4-wk of washout. HUCB-MNCs restored diminished intracavernosal pressure responses, acetylcholine-, sodium nitroprusside-, sildenafil-induced relaxations, and increased phenylephrine and electrical field stimulation (EFS)-induced contractions. Decreased EFS-induced relaxations in dutasteride-treated groups were not restored by HUCB-MNCs. Increased PDE5 and reduced nNOS expressions in dutasteride groups were restored by HUCB-MNCs in the 12-week dutasteride group. eNOS and HIF-1α protein expression and serum total and free testosterone levels were similar among groups. HUCB-MNCs reversed the decreased smooth muscle/collagenratio in dutasteride-treated tissues. There was a significant increase in PDE5 and HIF-1α staining in 8-week dutasteride animals. This study demonstrates the corrective potential of HUCB-MNCs on some persistent structural and functional deterioration caused by 5ARI treatment in rats, which may encourage further evaluation of HUCB-MNCs in men with PPED. Therapeutic application of intracavernosal HUCB-MNCs is a novel approach for the rat model of post-5ARI ED. Lack of serum and tissue dihydrotestosterone measurements, vehicle injections and characterization of the cells remain limitations of our study. The persistent ED after prolonged administration of dutasteride in rats is reversed by HUCB-MNC treatment, which holds promise as a realistic therapeutic modality for this type of ED. Oztekin CV, Yilmaz-Oral D, Kaya-Sezginer E, etal. Beneficial Effects of Human Umbilical Cord Blood Mononuclear Cells on Persistent Erectile Dysfunction After Treatment of 5-Alpha Reductase Inhibitor in Rats. J Sex Med 2021;18:889-899.

Testosterone positively regulates functional responses and nitric oxide expression in the isolated human corpus cavernosum.

Testosterone (T) deficiency is associated with erectile dysfunction (ED). The relaxant response of T on the corporal smooth muscle through a non-genomic pathwayhas been reported; however, the in vitro modulating effects ofTon human corpus cavernosum (HCC) have not been studied. To compare the effects of various concentrations of T on nitric oxide (NO)-dependent and nitric oxide-independent relaxation in organ bath studies and elucidate its mode of action, specifically targeting the cavernous NO/cyclic guanosine monophosphate (cGMP) pathway. Human corpus cavernosum (HCC) samples were obtained from men undergoing penile prosthesis implantation (n=9). After phenylephrine (Phe) precontraction, the effects of various relaxant drugs of HCC strips were performed using organ bath at low (150ng/dL), eugonadal (400ng/dL), and hypergonadal (600ng/dL) T concentrations. The penile tissue measurements of endothelial nitric oxide synthase (eNOS), neuronal (n)NOS, and phosphodiesterase type 5 (PDE5) were evaluated via immunostaining, Western blot, cGMP and nitrite/nitrate (NOx) assays. Relaxation responses to ACh and EFS in isolated HCC strips were significantly increased at all T levels compared with untreated tissues. The sildenafil-induced relaxant response was significantly increased at both eugonadal and hypergonadal T levels. Normal and high levels of T are accompanied by increased eNOS, nNOS, cGMP, and NOx levels, along with reduced PDE5 protein expression. This study reveals an important role of short-term and modulatory effects of different concentrations of T in HCC. T positively regulates functional activities, inhibition of PDE5 expression, and formation of cGMP and NOx in HCC. These results demonstrate that T indirectly contributes to HCC relaxation via downstream effects on nNOS, eNOS, and cGMP and by inhibiting PDE5. This action provides a rationale for normalizing T levels in hypogonadal men with ED, especially when PDE5 inhibitors are ineffective. T replacement therapy may improve erectile function by modulating endothelial function in hypogonadal men.

Discovery of Novel Agents on Spindle Assembly Checkpoint to Sensitize Vinorelbine-Induced Mitotic Cell Death Against Human Non-Small Cell Lung Cancers.

Non-small cell lung cancer (NSCLC) accounts about 80% of all lung cancers. More than two-thirds of NSCLC patients have inoperable, locally advanced or metastatic tumors. Non-toxic agents that synergistically potentiate cancer-killing activities of chemotherapeutic drugs are in high demand. YL-9 was a novel and non-cytotoxic compound with the structure related to sildenafil but showing much less activity against phosphodiesterase type 5 (PDE5). NCI-H460, an NSCLC cell line with low PDE5 expression, was used as the cell model. YL-9 synergistically potentiated vinorelbine-induced anti-proliferative and apoptotic effects in NCI-H460 cells. Vinorelbine induced tubulin acetylation and Bub1-related kinase (BUBR1) phosphorylation, a necessary component in spindle assembly checkpoint. These effects, as well as BUBR1 cleavage, were substantially enhanced in co-treatment with YL-9. Several mitotic arrest signals were enhanced under combinatory treatment of vinorelbine and YL-9, including an increase of mitotic spindle abnormalities, increased cyclin B1 expression, B-cell lymphoma 2 (Bcl-2) phosphorylation and increased phosphoproteins. Moreover, YL-9 also displayed synergistic activity in combining with vinorelbine to induce apoptosis in A549 cells which express PDE5. In conclusion. the data suggest that YL-9 is a novel agent that synergistically amplifies vinorelbine-induced NSCLC apoptosis through activation of spindle assembly checkpoint and increased mitotic arrest of the cell cycle. YL-9 shows the potential for further development in combinatory treatment against NSCLC.

Higher expression of phosphodiesterase type 5 in the anterior fibromuscular stroma of the human prostate.

To examine phosphodiesterase type 5 (PDE5) expression in the anterior fibromuscular stroma (AFMS) of the prostate. Although PDE5 expression was identified in the human prostate, differences in PDE5 expression in intra-prostatic regions are unknown. The AFMS in the prostate has peculiar innervations that could contribute to voiding function. Here, we examined regional differences in PDE5 expression in the prostate with special reference to the AFMS. A total 18 human prostate and bladder specimens were obtained. Tissue specimens were processed by hematoxylin-eosin (H&E) staining and immunohistochemistry for PDE5. Immunoreactivity with PDE5 was evaluated using computer-assisted image analysis in the following regions: the AFMS, bladder neck, stromal hyperplasia in the transition zone, glandular hyperplasia in the transition zone (TZ gland), and the peripheral zone (PZ). The correlation between PDE5 expression in the AFMS and clinical data was analysed. Image analysis revealed that the median ratio of the PDE5-immunoreactive area to smooth muscle area by H&E staining was 74.7% in the AFMS. There was significantly higher PDE5 expression in the AFMS than in the TZ gland (p = 0.034) and PZ (p = 0.002). PDE5 expression in the AFMS was not significantly correlated with age, prostate volume, transition zone volume, or transition zone index. However, older men had a tendency to have higher PDE5 expression in the AFMS. We found higher PDE5 expression in the AFMS compared with other prostatic regions, which suggested that the AFMS is a target region of PDE5 inhibitors in the prostate.

P66Shc Deletion Ameliorates Oxidative Stress and Cardiac Dysfunction in Pressure Overload-Induced Heart Failure

p66Shc is a redox enzyme that plays an important role in the response of oxidative stress and the p53-dependent apoptosis. The expression level of p66Shc has a negative correlation with the resistance of oxidative stress in vivo and in vitro. We aim to demonstrate the function of p66Shc in pressure overload-induced heart failure. The pressure overload-induced heart failure was induced in mice by transverse aortic constriction (TAC). Cardiac dysfunction was shown by transthoracic echocardiography. Western blot was used to check the protein levels of phosphodiesterase type 5 (PDE5) and ventricular oxidative stress markers. Superoxide dismutase (SOD) mimetic M40401 and PDE5 inhibitor sildenafil were used in the treatment of mice. The deletion of p66Shc attenuated cardiac dysfunction and oxidative stress in pressure overload-induced heart failure. p66Shc deletion also decreased the expression of ventricular oxidative stress markers and enhanced PKG signaling by promoting the expression of PDE5. M40401 and sildenafil attenuated the TAC-induced cardiac dysfunction and oxidative stress in p66Shc overexpression mice. Our findings suggest that p66Shc participates in the regulation of cardiac dysfunction and oxidative stress in TAC-derived pressure overload-induced heart failure in mice, and SOD and PDE5 are molecules downstream of p66Shc in this regulatory process.

Optimization of Extraction Technology of Majun Mupakhi Ela and its Effect on Hydrocortisone-induced Kidney Yang Deficiency in Mice

We used Box-Behnken design-based (BBD) response surface methodology (RSM) in this research to optimize the extraction process of Traditional medicine Majun Mupakhi Ela (MME) and evaluate its effect on hydrocortisone-induced kidney yang deficiency. Three independent parameters were applied to evaluate the maximum phosphodiesterase type 5 (PDE5) inhibition activity of MME extracts in vitro. The optimal processing conditions (extraction time 2 h, solid-liquid ratio 1:16, extraction once) gave a maximum PDE5 inhibition rate of 84.10%, flavonoid content of 0.49 mg/ml, icariin content of 0.028 mg/ml and targeted extraction yield of 26.50%. In animal experiments, MME extracts significantly increased the adrenal mass index, semen weight index, preputial gland weight index, and penis weight index in mice; in the middle and high dose group, the level of serum testosterone increased by 7664.29% and 14207.14% respectively, compared with the model group, and the level of PDE5 decreased by 67.22% and 74.69% respectively compared with the control group. These results indicate that MME has a significant positive effect on the hypothalamus-pituitary-gonadal axis, improve mating ability and not only has inhibits PDE5 activity but also significantly inhibits the expression of PDE5 in penile tissues, potential to become erectile dysfunction (ED) therapies for the clinical management of patients with kidney yang deficiency.

Intravascular Hemolysis Leads to Priapism Phenotype: Experimental Evidence

Introduction: Sickle cell disease (SCD) men display priapism. Berkeley and Townes SCD transgenic mice also display priapism phenotype associated with increased corpus cavernosum relaxation. Primary mechanism underlying priapism appears to be due to reduced nitric oxide bioavailability, which leads to downregulation of phosphodiesterase type 5 (PDE5) in the penis. Previous clinical studies reported that priapism is associated with increased intravascular hemolysis at SCD. Intravascular hemolysis releases components that impair nitric oxide signaling, such as hemoglobin, asymmetric dimethylarginine and arginase-1. It is well-established that nitric oxide scavenging by cell-free hemoglobin contributes to vascular dysfunction. However, no detailed study has investigated the role of intravascular hemolysis on pathophysiological alterations of erectile function. Since intravascular hemolysis can influence the biological activity of nitric oxide pathway, we hypothesized that excess concentration of plasma hemoglobin may contribute to priapism in SCD. Therefore, in the present study, we have used a murine model of intravascular hemolysis induced by phenylhydrazine (PHZ) to evaluate the effects of intravascular hemolysis on the nitric oxide-mediated corpus cavernosum relaxations.Methods: Intravascular hemolysis was induced in C57BL/6 mice by intraperitoneal injection of PHZ. C57BL/6 mice were injected with PHZ at 50 mg/Kg and then reinjected with 50 mg/Kg 8h later. Mice were killed in isoflurane chamber at 2 days after the first injection with PHZ. Strips of corpus cavernosum were mounted in isolated organ baths, and the relaxing responses to acetylcholine (ACh; endothelium-dependent response) and sodium nitroprusside (SNP; endothelium-independent response), as well as electrical-field stimulation (EFS; nitrergic relaxation) were obtained in corpus cavernosum strips precontracted with the α1-adrenergic receptor agonist phenylephrine (10 µM).Results: Plasma hemoglobin was significantly higher in PHZ compared to control mice (11±1 and 4±1 µM, respectively; P<0.001), which demonstrate the increase in the rate of intravascular hemolysis in PHZ-treated animals. The cumulative addition of ACh (0.001-10 µM) produced concentration-dependent corpus cavernosum relaxations in both groups, but maximal relaxations were significantly higher in PHZ (59±4 %; P<0.05, n=7) compared to control mice (45±4%). Likewise, SNP (0.01-10 µM) produced concentration-dependent corpus cavernosum relaxations, but, again, the maximal relaxations induced by this agent were significantly higher in PHZ (87±2%, P<0.01) compared to the control group (74±1%, n=7). Similarly, the nitrergic relaxations caused by EFS were also significantly higher (P<0.05) in PHZ mice compared to control mice (32 Hz: 79±2 and 66± 1%, respectively; n=5).Conclusion: Our study shows that intravascular hemolysis leads to increased nitric oxide-mediated cavernosal relaxations, indicating that PHZ mice exhibit priapism phenotype. It is likely that hemoglobin act to inactivate nitric oxide, thus reducing the amount of bioavailable nitric oxide, which may lead to downregulation of PDE5 in the penis. One may speculate, therefore, that the reduced degradation of cGMP by PDE5 in PHZ mice may lead to the enhancement of nitric oxide-mediated cavernosal relaxations. To confirm this hypothesis, we will evaluate the expression of PDE5 and cGMP levels in PHZ mice penises.Financial Support: FAPESP. DisclosuresNo relevant conflicts of interest to declare.

Analysis of short-term treatment with the phosphodiesterase type 5 inhibitor tadalafil on long bone development in young rats

Cyclic GMP (cGMP) is an important intracellular regulator of endochondral bone growth and skeletal remodeling. Tadalafil, an inhibitor of the phosphodiesterase (PDE) type 5 (PDE5) that specifically hydrolyzes cGMP, is increasingly used to treat children with pulmonary arterial hypertension (PAH), but the effect of tadalafil on bone growth and strength has not been previously investigated. In this study, we first analyzed the expression of transcripts encoding PDEs in primary cultures of chondrocytes from newborn rat epiphyses. We detected robust expression of PDE5 as the major phosphodiesterase hydrolyzing cGMP. Time-course experiments showed that C-type natriuretic peptide increased intracellular levels of cGMP in primary chondrocytes with a peak at 2 min, and in the presence of tadalafil the peak level of intracellular cGMP was 37% greater ( P < 0.01) and the decline was significantly attenuated. Next, we treated 1-mo-old Sprague Dawley rats with vehicle or tadalafil for 3 wk. Although 10 mg·kg-1·day-1 tadalafil led to a significant 52% ( P < 0.01) increase in tissue levels of cGMP and a 9% reduction ( P < 0.01) in bodyweight gain, it did not alter long bone length, cortical or trabecular bone properties, and histological features. In conclusion, our results indicate that PDE5 is highly expressed in growth plate chondrocytes, and short-term tadalafil treatment of growing rats at doses comparable to those used in children with PAH has neither obvious beneficial effect on long bone growth nor any observable adverse effect on growth plate structure and trabecular and cortical bone structure.

An immunohistochemical and ultrastructural analysis of the retina in tadalafil (Cialis) treated rats

Tadalafil (Cialis) is one of the most commonly used phosphodiesterase type5 (PDE5) inhibitors. This work aimed to analyze the histological and ultrastructural changes provoked by chronic tadalafil administration in the rat retina, correlate between such changes and PDE5 immunoexpression and to evaluate the possible reversibility of these changes. Thirty Sprague Dawley male rats were randomly distributed into 3 groups. Control group; given 1 ml distilled water daily for 6 weeks. Tadalafil group; given tadalafil in a daily dose of 2.6 mg/kg for 6 weeks. Withdrawal group; given tadalafil 2.6 mg/kg daily for 6 week followed by a withdrawal period of 4 weeks. Retinal specimens were prepared for histological, ultrastructural and immunohistochemical study using anti-PDE5 and anti-Bcl-2 antibodies. Morphometric and statistical studies were performed. Tadalafil group displayed a significant reduction in retinal thickness, diminished cell population of outer and inner nuclear layers, dilated blood capillaries and a significant decline in the number of ganglion cells. Significant reductions of both PDE5 and Bcl-2 immunoexpression were observed. At the ultrastructural level, the photoreceptors showed spacing of outer segments and disorganized membranous discs. Retinal neurons showed ultrastructural degenerative changes in the form of shrunken irregular nuclei, dilated rER, and disrupted mitochondria. Withdrawal group revealed preservation of histological structure and partial restoration of retinal thickness, retinal cells ultrastructure, and PDE5 and Bcl-2 immunoexpressions. In conclusion, chronic use of tadalafil could induce reversible apoptotic and degenerative changes in retinal neurons due to its inhibitory effect on PDE5 expression which affects the metabolism and viability of retinal cells.

Tadalafil induces antiproliferation, apoptosis, and phosphodiesterase type 5 downregulation in idiopathic pulmonary arterial hypertension in vitro

Idiopathic pulmonary arterial hypertension (IPAH) is a fatal disease of the pulmonary artery resulting from a currently unidentified etiology. IPAH is pathologically characterized as sustained vasoconstriction and vascular remodeling of the pulmonary artery. Vascular remodeling is mediated by enhanced proliferation and reduced apoptosis in pulmonary arterial smooth muscle cells (PASMCs). Based on its pathological mechanism, specific phosphodiesterase type 5 (PDE5) inhibitors have been used in the treatment of IPAH. In addition to sildenafil, tadalafil has been approved for the treatment of IPAH. However, the effects of tadalafil on excessive proliferation of IPAH-PASMCs currently remain unknown. In the present study, the in vitro pharmacological profiles of tadalafil for cell proliferation and apoptosis were assessed in IPAH-PASMCs using MTT, BrdU incorporation, and caspase 3/7 assays. Expression analyses revealed that PDE5 mRNA and protein expression levels were markedly higher in IPAH-PASMCs than in normal-PASMCs. The treatment with tadalafil inhibited the excessive proliferation of IPAH-PASMCs in a concentration-dependent manner with an IC50 value of 4.5μM. On the other hand, tadalafil (0.03–100μM) did not affect cell growth of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). In addition, tadalafil induced apoptosis in IPAH-PASMCs. The antiproliferative and apoptotic effects of tadalafil were markedly stronger than those of sildenafil and vardenafil. The upregulated expression of PDE5 in IPAH-PASMCs was significantly attenuated by a long-term treatment with tadalafil. Taken together, these results indicate that tadalafil attenuates vascular remodeling by inhibiting cell proliferation, promoting apoptosis, and downregulating PDE5 in IPAH-PASMCs, thereby ameliorating IPAH.

Carbon-11 and Fluorine-18 Radiolabeled Pyridopyrazinone Derivatives for Positron Emission Tomography (PET) Imaging of Phosphodiesterase-5 (PDE5).

The cyclic guanosine monophosphate (cGMP) specific phosphodiesterase type 5 (PDE5) plays an important role in various pathologies including pulmonary arterial hypertension and cardiomyopathy. PDE5 represents an important therapeutic and/or prognostic target, but noninvasive assessment of PDE5 expression is lacking. The purpose of this study was to develop and evaluate pyridopyrazinone derivatives labeled with carbon-11 or fluorine-18 as PDE5-specific PET tracers. In biodistribution studies, highest PDE5-specific retention was observed for [11C]-12 and [18F]-17 in the lungs of wild-type mice and in the myocardium of transgenic mice with cardiomyocyte-specific PDE5 overexpression at 30 min postinjection. In vivo dynamic microPET images in rats revealed that both tracers crossed the blood-brain barrier but brain retention was not PDE5-specific. Both [11C]-12 and [18F]-17 showed specific binding to PDE5 in myocardium of transgenic mice; however [18F]-17 showed significantly higher PDE5-specific inhibitable binding than [11C]-12.

S-nitrosylation of PDE5 increases its ubiquitin–proteasomal degradation

Phosphodiesterase type 5 (PDE5) expression is upregulated in human failing heart, and overexpression of PDE5 in transgenic mice exacerbates stress-induced left-ventricular dysfunction, suggesting that increased PDE5 expression might contribute to the development of congestive heart failure. However, the underlying mechanisms for increased PDE5 expression are not totally understood. In the present study, we found that PDE5 activity and expression were regulated by S-nitrosylation, a covalent modification of cysteine residues by nitric oxide (NO). S-nitrosylation of PDE5 occurs at Cys220, which is located in the GAFA domain. Upon S-nitrosylation, PDE5 exhibits reduced activity and degradation via the ubiquitin–proteasome system. The decrease in PDE5 expression induced by NO could be blunted by mutation of Cys220 or the phosphorylation site of PDE5 (S102), as well as by pretreatment with H2O2. Conversely, decreased NO bioavailability by nitric oxide synthase (NOS) inhibitors or knockout of NOS3 increased PDE5 expression in cardiomyocytes. Collectively, to the best of our knowledge, our data demonstrate for the first time that S-nitrosylation is one of the mechanisms for PDE5 degradation. This novel regulatory mechanism probably accounts for the increase in PDE5 in the failing heart and other diseases in which NO bioavailability is decreased by oxidative stress.

MP31-06 UPREGULATION OF PHOSPHODIESTERASE TYPE 5 IN THE HYPERPLASTIC PROSTATE: A RAT MODEL STUDY

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 2015MP31-06 UPREGULATION OF PHOSPHODIESTERASE TYPE 5 IN THE HYPERPLASTIC PROSTATE: A RAT MODEL STUDY Wenhao Zhang, Ping Chen, Devendra Singh Negi, Zhuo Li, Keke Zhao, Qi Mao, and Xinhua Zhang Wenhao ZhangWenhao Zhang More articles by this author , Ping ChenPing Chen More articles by this author , Devendra Singh NegiDevendra Singh Negi More articles by this author , Zhuo LiZhuo Li More articles by this author , Keke ZhaoKeke Zhao More articles by this author , Qi MaoQi Mao More articles by this author , and Xinhua ZhangXinhua Zhang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1361AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Both erectile dysfunction (ED) and lower urinary tract symptoms (LUTS)/ benign prostatic hyperplasia (BPH) are quite common for aging males. Phosphodiesterase type 5 inhibitors (PDE5-Is) have been proven as an effective pharmacotherapy for men suffering from ED. Numerous clinical trials have also confirmed the efficacy of PDE5-Is for LUTS/BPH, while tadalafil was recently licensed in USA and in European Union for treating LUTS/BPH with or without ED. However, the mechanisms of PDE5-Is in treating LUTS/BPH are still unclear. In this study we developed a rat model of BPH and the major contraction and relaxation molecules in the prostate were investigated with phosphodiesterase type 5 (PDE5) emphasized. METHODS 250g-300g male Wistar rats were divided into two groups: group 1 (n=16) was treated with 0.1 ml sesame oil; group 2 (n=16) was treated with subcutaneous injection of testosterone propionate (TP) (2 mg/d) for 30 days. Rats were weighed and sacrificed under anesthesia on day 31. The whole ventral prostatic lobes were quickly removed and weighed. PDE5, nitric oxide synthase isoforms (nNOS and eNOS) and adrenoreceptor subtypes (α1a,α1b and α1d) were determined with real time RT-PCR. PDE5 was further analyzed with western-blot and histological examination. Serum testosterone (T) was measured with ELISA. RESULTS BPH model was validated with significantly enlarged prostate and H-E stain. BPH rats group also showed a significant increased serum T level. PDE5 localized mainly in fibromuscular stroma cells as well as in endothelial and smooth muscle cells of blood vessels in prostate. BPH upregulated PDE5 expression by two fold at the gene level and approximately threefold at transcriptional level. Real time RT-PCR also showed that α1a, α1d and nNOS were augmented significantly in BPH group but with no change in the level of eNOS and α1b between BPH and control group. CONCLUSIONS Our novel data showed a higher expression of PDE5 in the BPH group than in controls. The increased expression of PDE5 mRNA and protein in BPH rat prostate could explain the effectiveness and possible mechanism of PDE5-Is in treating LUTS/BPH. Fibromuscular stroma cells as well as endothelial and smooth muscle cells of blood vessels could be the main target tissue of PDE5-Is in the prostate. However, further functional studies are required. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e357 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Wenhao Zhang More articles by this author Ping Chen More articles by this author Devendra Singh Negi More articles by this author Zhuo Li More articles by this author Keke Zhao More articles by this author Qi Mao More articles by this author Xinhua Zhang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Reduction of obstruction related bladder overactivity by the guanylyl cyclase modulators BAY 41-2272 and BAY 60-2770 alone or in combination with a phosphodiesterase type 5 inhibitor.

To assess the urodynamic effects of soluble guanylyl cyclase (sGC) stimulator, BAY 41-2272, and activator, BAY 60-2770, (which both are able to induce cGMP synthesis even in the absence of nitric oxide (NO)) alone or in combination with a phosphodiesterase type 5 (PDE5) inhibitor, vardenafil, in a model of partial urethral obstruction (PUO) induced bladder overactivity (BO). Fifty-six male Sprague-Dawley rats were used, 31 of them underwent PUO. Fourteen rats were used for Western blots to assess PDE5 and sGC expression. For drug evaluation cystometry without anesthesia was performed three days following bladder catheterization. Obstructed rats showed higher micturition frequency and bladder pressures than non-obstructed animals (Intermicturition Interval, IMI, 2.28 ± 0.55 vs. 3.60 ± 0.60 min (± standard deviation, SD); maximum micturition pressure, MMP, 70.1 ± 8.0 vs. 48.8 ± 7.2 cmH2O; both P < 0.05). In obstructed rats vardenafil, BAY 41-2272, and BAY 60-2770 increased IMI (2.77 ± 1.12, 2.62 ± 0.52, and 3.22 ± 1.04 min; all P < 0.05) and decreased MMP (54.4 ± 2.8, 61.5 ± 11.3, and 51.2 ± 6.3 cmH2O; all P < 0.05). When vardenafil was given following BAY 41-2272 or BAY 60-2770 no further urodynamic effects were observed. PDE5 as well as sGC protein expression was reduced in obstructed bladder tissue. Targeting sGC via stimulators or activators, which increase the levels of cGMP independent of endogenous NO, is as effective as vardenafil to reduce urodynamic signs of BO. Targeting the NO/cGMP pathway via compounds acting on sGC might become a new approach to treat BO.

Platelet-derived growth factor regulation of type-5 phosphodiesterase in human and rat penile smooth muscle cells.

Relaxation of cavernous smooth muscle cells (SMCs) is a key component in the control of the erectile mechanism. SMCs can switch their phenotype from a contractile differentiated state to a proliferative and dedifferentiated state in response to a change of local environmental stimuli. Proliferation and contraction are both regulated by the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are degraded by phosphodiesterases (PDEs). The most abundant PDE present in corpora cavernosa is the electrolytic cGMP-specific phosphodiesterase type 5 (PDE5). We investigated the cellular localization of PDE5 in in vitro cultured corpora cavernosa cells and the effect of mitogenic stimulation on PDE5 expression. Biochemical ad molecular techniques on cultured SMCs from human and rat penis. We studied the ability of the quiescent SMC phenotype vs. the proliferating phenotype in modulation of PDE5 expression. We demonstrated that PDE5 is localized in the cytoplasm, in the perinuclear area, and in discrete cytoplasmic foci. As previously demonstrated in human myometrial cells, the cytoplasmic foci may correspond to centrosomes. In corpora cavernosa, PDE5 protein levels are strongly regulated by the mitotic activity of the SMCs, as they were increased in quiescent cultures. In contrast, treatment with platelet-derived grow factor (PDGF), one of the most powerful mitogenic factors for SMCs, reduces the expression of PDE5 after 24 hours of treatment. We found that PDGF treatment downregulates PDE5 expression in proliferating SMCs, suggesting that PDE5 may represent one of the markers of the contractile phenotype of the SMCs of corpora cavernosa.

Sildenafil for Noncompaction Cardiomyopathy Treatment in a Child: Case Report

Congestive heart failure (CHF) occurs when the heart can no longer maintain the baseline metabolic demand triggered by physiological venous pressure. In the pediatric population, it corresponds to a complex entity with multiple etiologies, depending on the age group assessed, being mainly associated with congenital structural malformations, cardiomyopathies, or secondary to arrhythmogenic, infectious, ischemic, toxic or infiltrative events1. The incidence of CHF in children also varies according to the underlying condition, ranging from 15% to 25% in patients with congenital heart defects. In structurally normal hearts, cardiomyopathy is the major factor associated with CHF, with a 40% incidence of CHF reported in those patients1. Isolated noncompaction cardiomyopathy is a rare disease, whose incidence in the general population ranges from 0.014% to 1.3%. It might result from a failure in compaction of the ventricular myocardium between weeks 5 and 8 of the embryonic life, leading to the persistence of myocardial trabeculations and deep recesses, which communicate with the ventricular cavity, generating myocardial thickening in two distinct layers (compacted and noncompacted). Initially reported in the pediatric population or with a congenital cardiopathy, it also affects adults with no other heart disease2,3. This failure in the regression of embryonic sinusoids is postulated to be due to the extremely high pressures that the ventricles undergo in that developmental period. However, the literature has reported a genetic aspect emphasizing the relationship of the disease with different genes, such as the mutation of gene G4.5 in families with severe infantile noncompaction cardiomyopathy, mutations P121L, CYPHER/ZASP, E101K, and a locus containing the disease gene in chromosome 11p153. The major complications of patients with noncompaction cardiomyopathy are pulmonary thromboembolism, arrhythmias and CHF3. In addition to the usual treatment of CHF, new classes of drugs, such as the phosphodiesterase type 5 (PDE-5) inhibitors, have been studied4. They are currently used for the treatment of erectile dysfunction and pulmonary arterial hypertension, and began to be studied for the treatment of CHF after the demonstration of higher PDE-5 expression in myocytes and vascular muscle cells, as well as of markers of oxidative stress in patients with CHF5. We report, for the first time, the clinical and laboratory findings of a patient with noncompaction cardiomyopathy after the addition of sildenafil (PDE-5 inhibitor) to the usual treatment.