Human bladder as a novel target for vitamin D receptor ligands.

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.

BXL-628, a vitamin D receptor agonist effective in benign prostatic hyperplasia treatment, prevents RhoA activation and inhibits RhoA/Rho kinase signaling in rat and human bladder.

BACKGROUND: BXL-628 is a calcitriol analog shown to decrease prostate growth in preclinical and clinical studies. BPH symptoms are generated not only by prostate overgrowth but also by bladder overactivity, resulting from an increased RhoA/Rho-kinase signaling. Because bladder smooth muscle cells express VDR, we studied effects of BXL-628 on this pathway. METHODS: RhoA and Rho-kinase gene expression and functional activity were studied in rat and human bladder smooth muscle by real-time RT-PCR, immuno-kinase assays, western blot analysis, confocal microscopy, in vitro contractility, and cell migration. RESULTS: In bladder smooth muscle, carbachol responsiveness was delayed and Rho-kinase activity reduced by BXL-628 treatment because of impaired RhoA membrane translocation and activation. Accordingly, RhoA-mediated biological functions, such as cell migration and cytoskeleton remodeling were also inhibited by BXL-628. CONCLUSIONS: BXL-628 inhibits RhoA/Rho-kinase signaling, a calcium sensitizing pathway, suggesting its possible clinical use in the treatment of altered bladder contractility often associated with BPH-induced lower urinary tract symptoms.

Testosterone restores diabetes-induced erectile dysfunction and sildenafil responsiveness in two distinct animal models of chemical diabetes.

INTRODUCTION: Hypogonadism is often associated with diabetes and both conditions represent major risk factors for erectile dysfunction (ED). AIM: To investigate the role of hypogonadism on phosphodiesterase type 5 (PDE5) expression and sildenafil responsiveness in diabetes. METHODS: Two different models of experimental diabetes were used: (i) alloxan-induced diabetic rabbit; and (ii) streptozotocin (STZ)-induced diabetic rat. In both experimental models, animals were separated into three groups: control, diabetic, diabetic supplemented with testosterone (T) enanthate. Rabbits were used for "in vitro" experiments. Conversely, each rats group was further subdivided: no further treatment or acute sildenafil dosing (25 mg/kg, 1 hour before "in vivo" electrical stimulation [ES]). MAIN OUTCOME MEASURE: Erectile capacity was evaluated either by "in vitro" contractility study (alloxan-induced diabetic rabbit) and "in vivo" evaluation of erectile response elicited by ES of cavernous nerve (STZ-induced diabetic rats). Also endothelial nitric oxide synthase, neural nitric oxide synthase (nNOS), and PDE5 protein (Western blot) and mRNA (quantitative real-time reverse transcriptase polymerase chain reaction [RT-PCR]) expression were measured in rat penile samples of each group. RESULTS: In both models, hypogonadism was observed, characterized by reduced T and atrophy of androgen-dependent accessory glands. T substitution completely reverted hypogonadism and diabetes-induced penile hyposensitivity to "in vitro" (acetylcholine, rabbit) or "in vivo" (ES, rat) relaxant stimuli, along with nNOS expression, which was reduced (P < 0.05) in STZ rats. In diabetic animals, T substitution reinstated sildenafil-induced enhancement of both "in vitro" nitric oxide donor (NCX 4040) relaxant effect (rabbit) and "in vivo" ES-induced erection (rat). PDE5 was reduced in diabetic STZ rats (P < 0.05) and normalized by T. In STZ rats, sodium nitroprusside (SNP) intracavernous injection induced a more sustained erection than in control rats, which was no further enhanced by sildenafil. T substitution normalized both hyper-responsiveness to SNP and sildenafil efficacy. CONCLUSION: In two models of diabetes T deficiency underlies biochemical alterations leading to ED. Normalizing T in diabetes restores nNOS and PDE5, and reinstates sensitivity to relaxant stimuli and responsiveness to sildenafil.

Effect of chronic tadalafil administration on penile hypoxia induced by cavernous neurotomy in the rat.

BACKGROUND: Numerous men develop postprostatectomy erectile dysfunction (PPED), due to surgery-related nervous damage. PPED is often refractory to phosphodiesterase type 5 (PDE5) inhibitors therapy. AIM: To verify whether chronic tadalafil (CT) preserves bilateral cavernous neurotomy (BCN)-induced penile damage and hypo-oxygenation. METHODS: In a rat model of BCN we evaluated in vitro and ex vivo effect of CT treatment (2 mg/kg, daily for 3 months). RESULTS: Bilateral cavernous neurotomy induced massive hypoxia and decreased muscle/fiber ratio, completely restored by CT. Hypersensitivity of hypoxic tissues to the relaxant effect of the endothelin type B receptor (ETB) agonist IRL-1620 was observed, along with ETB mRNA and protein overexpression. CT restored sensitivity to IRL-1620, and normalized ETB expression. Hypoxic penis showed increased sensitivity to the relaxant effect of the nitric oxide donor sodium nitroprusside (SNP), while acute tadalafil (100 nM) did not amplify the SNP effect. Accordingly, PDE5 mRNA and protein were reduced in BCN penile tissues. By restoring PDE5, CT decreased SNP-induced relaxation and rescued sensitivity to acute tadalafil. However, in hypoxic penis, CT normalizes neither acetylcholine hyporesponsiveness nor neuronal nitric oxide synthase-endothelial nitric oxide synthase expression. CONCLUSION: Chronic tadalafil restores some of the investigated BCN-induced alterations, including PDE5 and tadalafil efficacy.

Identification, characterization and biological activity of oxytocin receptor in the developing human penis.

Although abnormalities of the male external genitalia (MEG) are a relatively common problem, little is known concerning the molecular mechanisms that finely regulate penile development. We report here the expression of the oxytocin receptor (OTR) gene by real-time RT-PCR in human fetal tissues (11th-12th week of gestation), including the MEG. The developing penis expressed a very high level of OTR mRNA, only a half log(10) unit lower than fetal central nervous system, used as a positive control. The OTR protein is also highly expressed (western, immunohistochemistry and binding studies) and immunolocalized both in the mesenchymal body and in the surrounding blood capillaries, which will later constitute penile trabeculae and sinusoids. Binding studies using [125I]oxytocin antagonist ([125I]OTA) in cultured human fetal penile smooth muscle cells (hfPSMC) revealed the presence of specific OTR with a high capacity and affinity for oxytocin (OT) and for OTA. Increasing concentrations of OT dose-dependently induced intracellular Ca2+ mobilization. Furthermore, OTR mediated an increase in the proliferation and the migration of hfPSMC. In conclusion, we demonstrate that in the developing human MEG, OTR is highly expressed and might be involved in coordinating timely and appropriate proliferation and migration of the penile cells. Thus, OTR might represent an additional target for investigating human fetal MEG organogenesis.