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Biomedical subjects

Linda Vignozzi

Publications and source records attributed to Linda Vignozzi.

10 recordsLinked to original sources

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.

Animals↗

Characterization and functional role of androgen-dependent PDE5 activity in the bladder.

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.

3',5'-Cyclic-GMP Phosphodiesterases↗

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.

3',5'-Cyclic-GMP Phosphodiesterases↗

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.

Animals↗

Oxytocin mediates the estrogen-dependent contractile activity of endothelin-1 in human and rabbit epididymis.

Epididymis is a sex steroid (androgen + estrogen)-sensitive duct provided with spontaneous motility, allowing sperm transport. We previously reported that the oxytocin (OT) receptor (OTR) mediates an estrogen-dependent increase in epididymal contractility. Because endothelin (ET)-1 also regulates epididymal motility, we tested its sex steroid dependence in a rabbit model. We demonstrated that estrogens up-regulate responsiveness to ET-1, which is reduced by blocking aromatase activity (letrozole, 2.5 mg/kg) or by triptorelin (2.9 mg/kg)-induced hypogonadism, whereas it is fully restored by estradiol valerate (3.3 mg/kg weekly) but not by testosterone enanthate (30 mg/kg weekly). However, changing sex steroid milieu did not affect either ET-1, its receptor gene, or protein expression. Two structurally distinct OTR-antagonists [(d(CH2)5(1), Tyr(Me)(2), Orn(8))-OT and atosiban] almost completely abolished ET-1 contractility, without competing for [125I]ET-1 binding, suggesting that OT/OTR partially mediates ET-1 action. Immunohistochemical studies in human and rabbit epididymis demonstrated that both OT and its synthesis-associated protein, neurophysin I, are expressed in the epithelial cells facing the muscular layer, suggesting local OT production. Quantitative RT-PCR demonstrated a high abundance of OT transcripts in human epididymis. OT transcript was also originally detected and partially sequenced in rabbit epididymis. To verify whether ET-1 regulates OT release, we used rabbit epididymal epithelial cell cultures. These cells expressed a high density of [125I]ET-1 binding sites and responded to ET-1 with a dose-dependent OT release. Hence, we propose that an ET-1-induced OT/OTR system activation underlies the estrogen-dependent hyperresponsiveness to ET-1. These local sources might promote the spontaneous motility necessary for sperm transport.

Animals↗

Testosterone regulates PDE5 expression and in vivo responsiveness to tadalafil in rat corpus cavernosum.

OBJECTIVES: To investigate the effect of testosterone on PDE5 expression and PDE5 inhibitor tadalafil in vivo responsiveness in a rat model. METHODS: PDE5 expression was localized by immunohistochemistry in the rat corpus cavernosum (CC) and quantified by both real-time RT-PCR and Western blot analysis in several tissues. In the in vivo study, control, castrated and testosterone (T) supplemented castrated rats were treated with acute or chronic oral tadalafil. Erectile function was evaluated by monitoring intracavernous pressure (ICP) following electro-stimulation (ES) of the cavernous nerve and intracavernous injection of NO donor, sodium nitroprusside (SNP). RESULTS: Rat CC expressed the highest PDE5 mRNA level. PDE5 was specifically immunolocalized in endothelial and smooth muscle cells. Surgical castration induced a significant reduction of PDE5 gene and protein expression (p<0.05), and ES response at all stimulation frequencies (p<0.001). T supplementation completely restored PDE5 expression, erectile response to ES and responsiveness to PDE5 inhibitor. Both acute and chronic tadalafil treatment were ineffective in ameliorating the ES response in castrated rats. Injection of increasing concentrations of SNP in castrated rats resulted in a statistically significant increase in ICP/MAP ratio as that observed in intact rats. In addition, tadalafil did not amplify the SNP effect in castrated rats at all the doses tested (0.06-6 nmoles). CONCLUSIONS: Our findings demonstrate that testosterone positively regulates PDE5 expression and in vivo responsiveness to PDE5 inhibitor, tadalafil, in the rat CC.

3',5'-Cyclic-GMP Phosphodiesterases↗

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.

Calcium↗

Androgens regulate phosphodiesterase type 5 expression and functional activity in corpora cavernosa.

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.

3',5'-Cyclic-GMP Phosphodiesterases↗

Oxytocin receptor is expressed in the penis and mediates an estrogen-dependent smooth muscle contractility.

Oxytocin (OT) is released by the posterior pituitary during male orgasm and is supposed to participate in the ejaculatory process. We now report evidence demonstrating the presence of an OT receptor gene (real-time RT-PCR and Northern blot) and protein (immunohistochemistry, Western blot, and binding studies) expression in the rabbit and human corpus cavernosum (CC) and its possible involvement in postorgasmic penile detumescence. OT receptor is expressed in the penis at a concentration similar to that present in other portions of the male genital tract and mediates CC contractility. OT-induced CC contractility is clearly regulated by the changing sex steroid milieu. In fact, we found that in a rabbit model of hypogonadotropic hypogonadism (induced by a single administration of the long-acting GnRH agonist triptorelin pamoate, 2.9 mg/kg), OT responsiveness was strongly reduced and was completely restored by estradiol valerate (3.3 mg/kg weekly), but not by testosterone enanthate (30 mg/kg weekly). As we found that CC expresses both subtypes of estrogen receptors and P450 aromatase, we hypothesized a physiological role for endogenous estrogens in regulating OT responsiveness. We therefore treated adult rabbits with an aromatase inhibitor (letrozole, 2.5 mg/kg) or an antiestrogen (tamoxifen, 0.25 mg/kg) for 3 wk. Both treatments significantly reduced CC responsiveness to OT stimulation. In conclusion, these findings indicate that OT might participate in inducing postorgasmic penile flaccidity and suggest a new role for estrogens in the male: regulation of CC responsiveness to OT.

Animals↗

Estrogens, but not androgens, regulate expression and functional activity of oxytocin receptor in rabbit epididymis.

Previous binding and contractility studies indicate that oxytocin (OT) receptors are present in rabbit epididymis. To investigate the effect of changing endocrine milieu on OT responsiveness, we induced hypogonadism (hypo) in rabbits with a single administration of a long-acting GnRH analog, triptorelin, and we replaced hypogonadal rabbits with different sex steroids. After 2 months from triptorelin administration, testosterone (T) plasma levels were decreased and OT responsiveness abolished. Administration of T to hypo rabbits restored T plasma levels but not OT sensitivity. Because Western blot analysis indicated that both estrogen receptors and aromatase are expressed in the epididymis, we treated hypo rabbits with estradiol valerate (E2v). E2v not only completely restored OT responsiveness but also even amplified it. Accordingly, Northern and Western blot analysis indicated that both OT receptor gene and protein were strongly induced by E2v but not by T. Surprisingly, also the class I estrogen receptor antagonist, tamoxifen restored OT sensitivity in hypo rabbits. To verify whether endogenous estradiol is involved in the regulation of OT receptor responsiveness, we treated intact rabbits with an aromatase inhibitor, letrozole. Blocking aromatase activity almost completely abolished OT sensitivity. These findings suggest a new function of estrogens in the male: regulation of OT responsiveness in epididymis.

Androgens↗