By real-time RT-PCR and Western blot analysis, we found that phosphodiesterase type 5 (PDE5) mRNA and protein abundance was several fold higher in human male than in female reproductive tracts. The highest mRNA level (>1 x 10(7) molecules/microg total RNA) was detected in human corpora cavernosa (CC), where PDE5 protein was immunolocalized in both muscular and endothelial compartment. The possible role of androgens in regulating PDE5 expression was studied using a previously established rabbit model of hypogonadotropic hypogonadism. In this model, hypogonadism reduced, and testosterone (T) supplementation restored, CC PDE5 gene and protein expression. In addition, T supplementation completely rescued and even enhanced cyclic GMP conversion to metabolites, without changing IC(50) for sildenafil (IC(50) = 2.16 +/- 0.62 nm). In control CC strips, sildenafil dose-dependently increased relaxation induced by electrical field stimulation, with EC(50) = 3.42 +/- 1.7 nm. Hypogonadism reduced, and T increased, sildenafil effect on electrical field stimulation, again without changing their relative EC(50) values. CC sensitivity to the NO-donor NCX4040 was greater in hypogonadal rabbit strips than in control or T-treated counterparts. Moreover, sildenafil enhanced NCX4040 effect in eugonadal rabbit strips but not in hypogonadal ones. This suggests that androgens up-regulate PDE5 in rabbit penis. We also measured PDE5 gene expression and metabolic activity in human CC from male-to-female transsexual individuals, chronically treated with estrogens and cyproterone acetate. Comparing the observed values vs. eugonadal controls, PDE5 mRNA, protein, and functional activity were significantly reduced. In conclusion, we demonstrated, for the first time, that androgens positively regulate PDE5, thus providing a possible explanation about the highest abundance of this enzyme in male genital tract.

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Benign prostate hyperplasia is the most common disease in the aging male, often comorbid with erectile dysfunction. Phosphodiesterase type 5 (PDE5) inhibitors (sildenafil, tadalafil, and vardenafil) decrease lower urinary tract symptoms in patients with erectile dysfunction and BPH. We studied PDE5 expression and activity in the human bladder and PDE5i effects both in vitro (human and rat) and in vivo (rat). PDE5 is highly expressed in rat and human bladder and immunolocalized in vascular endothelium and muscle fibers. Sildenafil, tadalafil, and vardenafil blocked 70% of the total cGMP-catabolizing activity; vardenafil was the most potent (IC(50) = 0.3 nm). In human bladder cells and in rat strips, a PDE-resistant cGMP analog, SP-8-Br-PET-cGMPS, induced, respectively, a consistent antiproliferative and relaxant effect. In contrast, the nitric oxide donor sodium nitroprusside (SNP) was almost ineffective. However, blocking PDE5 with vardenafil increased SNP antiproliferative and relaxant activity up to the level observed with SP-8-Br-PET-cGMPS. We also found that castration decreased, and T supplementation restored, PDE5 gene expression in rat bladder. Accordingly, bladder strips from castrated rats were more sensitive to SNP-induced relaxation than strips from control or T-replaced rats, whereas in the presence of vardenafil, all groups showed the same SNP sensitivity. To discover whether vardenafil affects bladder activity in vivo, the rat bladder outlet obstruction model was used. Chronic treatment with 10 mg/kg.d vardenafil significantly reduced nonvoiding contractions (47%, P < 0.05 vs. placebo) up to tamsulosin level (51%). Overall, these results demonstrate that PDE5 regulates bladder smooth muscle tone, strongly limiting the nitric oxide/cGMP signaling, and that vardenafil, by blocking PDE5, may be a possible therapeutic option for bladder dysfunction by ameliorating irritative lower urinary tract symptoms.

Introduction Phosphodiesterase type 5 (PDE5) inhibitors ameliorate low urinary tract (LUT) symptoms in men with ED and symptomatic benign prostatic hyperplasia (BPH). PDE5 is highly expressed in rat and human bladder, where it regulates cyclic guanosine monophosphate (cGMP) degradation, muscle tone, and proliferation. Aim To investigate PDE5 tissue distribution and activity in human LUT tissues (urethra, prostate, and bladder). Main Outcome Measures PDE5 expression and activity were analyzed and compared within the same BPH patient in LUT tissues and in smooth muscle cells (SMCs) cultured from urethra, prostate, and bladder. Methods In LUT tissues, PDE5 was localized by immunohistochemistry and mRNA expression by quantitative real-time polymerase chain reaction. Proliferation assay was used as readout of PDE5 activity, evaluated as ability of vardenafil to increase the antiproliferative effect of different nitric oxide (NO)/cGMP pathway activators [the PDE5-resistant cGMP analog Sp-8-Br-PET-cGMPS, the NO donor sodium nitroprusside (SNP), and the soluble guanylate cyclase (sGC) stimulator BAY 41-8543]. Results In all the LUT tissues, PDE5 was immunolocalized in blood vessels and in muscular fibres, but not in epithelium. PDE5 mRNA expression was higher in urethra and bladder than in prostate SMC. The antiproliferative effect of Sp-8-Br-PET-cGMPS was similar in all LUT SMC. In prostatic SMC, SNP and BAY 41-8543 show a dose-dependent antiproliferative effect that resulted marginally enhanced by vardenafil. Conversely, in urethra and bladder SMC the antiproliferative effect of SNP and BAY 41-8543 was lower than in prostatic SMC, but it was significantly enhanced by vardenafil. In urethral and bladder cells vardenafil half-maximal response inhibiting concentration was in the subnanomolar range, whereas in prostate cells it resulted significantly higher. Conclusions The highest expression and biological activity of PDE5 was found in bladder. However, a consistent PDE5 expression and activity was also found in prostatic urethra. In contrast, the prostate gland showed the lowest PDE5 abundance and cultures derived from this tissue were less sensitive to vardenafil.

Human prostate is now considered a target for vitamin D receptor (VDR) ligands, such as BXL-628. Because BXL-628 inhibited prostate growth without interfering with androgen signaling, it represents a new option for benign prostate hyperplasia (BPH) therapy. However, BPH symptoms are related not only to prostate size, but also to compensatory bladder hypertrophy and eventual overactivity. We now report that human bladder expresses VDR (determined by real-time PCR immunohistochemistry and Western blot) and responds to VDR agonists, such as the natural ligand, calcitriol, and its synthetic and less hypercalcemic derivative, BXL-628. Experiments were conducted with stromal cells derived from human bladder neck obtained at surgery from BPH patients. BXL-628 counteracted keratinocyte growth factor (KGF) and androgen-induced cell proliferation and stimulated apoptosis with a parallel reduced expression of the survival oncoprotein Bcl-2. Prolonged serum starvation time-dependently pushed bladder stromal cells to express activated myofibroblast markers, such as desmin and smoothelin, without changing other contractile-related proteins and intermediate filaments, such as vimentin. Chronic exposure to BXL-628 prevented starvation-induced cell phenotype modification. Because hypertrophy and starvation-induced bladder remodeling are supposed to underlie bladder overactivity, it is possible that BXL-628 might be helpful in reducing not only cumbersome symptoms related to prostate overgrowth, but also those related to bladder irritation.

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Telocytes are a recently described stromal cell type widely distributed in various organs including the female and male reproductive systems. This study was aimed to investigate for the first time the existence, distribution and characteristics of telocytes in normal human testis by an integrated morphological approach (immunohistochemistry, immunofluorescence and transmission electron microscopy). We found that telocytes displaying typical long and moniliform prolongations and coexpressing CD34 and PDGFRα formed networks in the outer layer of peritubular tissue and around Leydig cells and vessels in the intertubular stroma. Testicular telocytes were immunophenotypically negative for CD31, c-kit/CD117 as well as α-SMA, thus making them clearly distinguishable from myoid cells/myofibroblasts located in the inner layer of peritubular tissue. Transmission electron microscopy confirmed the presence of cells ultrastructurally identifiable as telocytes (i.e. cells with telopodes alternating podomers and podoms) in the aforementioned locations. Intercellular contacts between neighboring telocytes and telopodes were observed throughout the testicular stromal compartment. Telopodes intimately surrounded and often established close contacts with peritubular myoid cells/myofibroblasts, Leydig cells and vessels. Extracellular vesicles were also frequently detected near telopodes. In summary, we demonstrated that telocytes are a previously neglected stromal component of human testis with potential implications in tissue homeostasis deserving further investigation.

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