Androgen deprivation therapy in prostate cancer: focusing on sexual side effects.

INTRODUCTION: Androgen deprivation therapy (ADT) is the cornerstone in the treatment of advanced and metastatic prostate cancer (PC). However, recent publications suggest that ADT may increase cardiovascular (CV) problems (morbidity and mortality), most probably because androgens regulate fat distribution, insulin sensitivity, and lipid metabolism. AIM: The aim of the present review is to analyze different modalities of ADT, emphasizing advantages and possible related adverse sexual events. METHOD: A systematic search of published evidence was performed using Medline (from 1969 to September 2011). MAIN OUTCOME MEASURE: The most important studies regarding the relationship among testosterone, PC, and the role of ADT were reviewed. RESULTS: There is unequivocal evidence that reducing androgen signaling to the hypogonadal range can reduce PC growth and patient symptoms; however, androgen dependency of prostate growth is evident only in the hypogonadal condition but not in the eugonadal state (the "saturation hypothesis"). There is clear evidence that ADT improves disease-free and overall survival only under two conditions: (i) in combination with primary radiation for locally advanced or high-risk diseases and (ii) as an adjuvant therapy for positive lymph node diseases after prostatectomy. On the other hand, it should be recognized that ADT can adversely affect not only traditional CV risk factor (including serum lipoproteins, insulin sensitivity, and obesity) but also sexual life, being associated with reduced libido and erectile, ejaculatory, and orgasmic disorders. CONCLUSIONS: Physicians should weigh the risk/benefit ratio before prescribing ADT.

Testosterone/estradiol ratio regulates NO-induced bladder relaxation and responsiveness to PDE5 inhibitors.

INTRODUCTION: The efficacy of phosphodiesterase type 5 inhibitors (PDE5i) in treating lower urinary tract symptoms is supported by the extremely high expression and activity of PDE5 in male bladder. Although bladder function regulation is similar among genders, no data are available on PDE5 expression and activity in female bladder. AIM: To investigate sex differences in PDE5 expression and biological activity in female bladder, as opposed to the male counterpart. MAIN OUTCOME MEASURE: Gene and protein expression and enzymatic activity of PDE5. METHODS: We studied gene and protein expression, and enzymatic activity of PDE5 in bladder of male and female rats. A subgroup of female rats was ovariectomized and alternatively replaced with estradiol (E2), progesterone, and testosterone (T) alone or in combination with letrozole to completely abrogate T-induced E formation. As a readout of PDE5 activity, we studied vardenafil efficacy in potentiating sodium nitroprusside (SNP)-induced relaxation in bladder of the different experimental groups. RESULTS: SNP was three-log unit less potent in relaxing the male bladder than the female one. On the contrary, the PDE5-resistant cyclic guanosine monophosphate (cGMP) analog (Bromo-ß-phenyl-1, N(2) -ethenoguanosine-3', 5'-cyclic monophosphorothioate, Sp-isomer [SP-8-Br-PET-cGMPS]) was equipotent in relaxing male and female bladder. Vardenafil was more effective in potentiating SNP-induced bladder relaxation in male than in female. Accordingly, the cGMP-hydrolyzing activity of PDE5 was higher in male vs. female homogenates. In ovariectomized female rats, with or without sex-steroid replacement, vardenafil activity in potentiating SNP-induced bladder relaxation was associated with an increased T/E2 ratio. In particular, masculinization of ovariectomized rats--by the administration of T + letrozole--dramatically increased vardenafil capacity to potentiate SNP-induced relaxation. CONCLUSION: In this study, we demonstrated that PDE5 activity is more pronounced in male as compared with female bladder and that T/E ratio positively regulates responsiveness to PDE5i, thus suggesting that male bladder is a more suitable target for PDE5i than the female counterpart.

Phosphodiesterase type 5 expression in human and rat lower urinary tract tissues and the effect of tadalafil on prostate gland oxygenation in spontaneously hypertensive rats.

INTRODUCTION: In humans, prostate phosphodiesterase type 5 inhibitors (PDE5) expression was prominently localized in the endothelial and smooth muscle cells of the vascular bed, suggesting a possible action of PDE5 inhibitors (PDE5i) on prostate blood flow. AIM: To investigate PDE5 expression in human and rat lower urinary tract (LUT) tissues, including vasculature, and determine the effects of PDE5 inhibition with tadalafil on prostatic blood perfusion. MAIN OUTCOME MEASURES: Human vesicular-deferential arteries (which originate from the inferior vesical artery, the main arterial source of blood supply to the bladder and prostate) were analyzed for PDE5 expression and activity. The effects of tadalafil on prostate oxygenation were studied in spontaneously hypertensive rats (SHR), characterized by ischemia/hypoxia of the genitourinary tract. METHODS: PDE5 expression was evaluated by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. SHR were treated with tadalafil (2 mg/kg/day) for 1, 7, or 28 days and compared with untreated SHR and the unaffected counterpart Wistar-Kyoto (WKY) rats. Prostate oxygenation was detected by Hypoxyprobe-1 and hypoxia markers (hypoxia-inducible factor-1α[HIF-1α] and endothelin-1 type B [ETB]) immunostaining. RESULTS: Human vesicular-deferential artery expressed high levels of PDE5, similar to corpora cavernosa, immunolocalized in the endothelial and smooth muscle layer. In these arteries, tadalafil inhibited cyclic guanosine monophosphate breakdown (half maximal inhibitory concentration (IC(50) ) in the low nanomolar range, as in corpora cavernosa) and increased the relaxant response to sodium nitroprusside. SHR prostate resulted markedly hypoxic (hypoxyprobe immunopositivity) and positive for HIF-1α and ETB, while tadalafil treatment restored oxygenation to WKY level at each time point. The mRNA expression of the HIF-1α target gene, BCL2/adenovirus E1B 19 kDa interacting protein 3, was significantly increased in SHR prostate and partially restored to WKY level by tadalafil. CONCLUSION: Human vesicular-deferential artery is characterized by a high expression and activity of PDE5, which was inhibited by tadalafil in vitro. In SHR, tadalafil increases prostate tissue oxygenation, thus suggesting a possible mechanism through which PDE5i exert beneficial effects on LUT symptoms.

Cavernous neurotomy in the rat is associated with the onset of an overt condition of hypogonadism.

BACKGROUND: Most men following radical retropubic prostatectomy (RRP) are afflicted by erectile dysfunction (ED). RRP-related ED occurs as a result of surgically elicited neuropraxia, leading to histological changes in the penis, including collagenization of smooth muscle and endothelial damage. AIM: To verify whether hypogonadism could contribute to the pathogenesis of RRP-ED. METHODS: Effects of testosterone (T), alone or in association with long-term tadalafil (Tad) treatment in a rat model of bilateral cavernous neurotomy (BCN). MAIN OUTCOME MEASURES: Penile tissues from rats were harvested for vasoreactivity studies 3 months post-BCN. Penile oxygenation was evaluated by hypoxyprobe immunostaining. Phosphodiesterase type 5 (PDE5), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) mRNA expression were quantified by Real Time quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: In BCN rats, we observed the onset of an overt condition of hypogonadism, characterized by reduced T plasma level, reduced ventral prostate weight, reduced testis function (including testis weight and number of Leydig cells), with an inadequate compensatory increase of luteinizing hormone. BCN induced massive penile hypoxia, decreased muscle/fiber ratio, nNOS, eNOS, PDE5 expression, increased sensitivity to the nitric oxide donor, sodium nitroprusside (SNP), and reduced the relaxant response to acetylcholine (Ach), as well as unresponsiveness to acute Tad dosing. In BCN rats, chronic Tad-administration normalizes penile oxygenation, smooth muscle loss, PDE5 expression, SNP sensitivity, and the responsiveness to the acute Tad administration. Chronic Tad treatment was ineffective in counteracting the reduction of nNOS and eNOS expression, along with Ach responsiveness. T supplementation, in combination with Tad, reverted some of the aforementioned alterations, restoring smooth muscle content, eNOS expression, as well as the relaxant response of penile strips to Ach, but not nNOS expression. CONCLUSION: BCN was associated with hypogonadism, probably of central origin. T supplementation in hypogonadal BCN rats ameliorates some aspects of BCN-induced ED, including collagenization of penile smooth muscle and endothelial dysfunction, except surgically induced altered nNOS expression.

Sex steroids and leptin regulate the "first Kiss" (KiSS 1/G-protein-coupled receptor 54 system) in human gonadotropin-releasing-hormone-secreting neuroblasts.

INTRODUCTION: The G-protein-coupled receptor 54 (GPR54) and its ligand kisspeptin, encoded by the KiSS-1 gene, have been involved in the molecular mechanisms underlying the reawakening of gonadotropin-releasing hormone (GnRH) neurons at puberty. GPR54 mutations cause hypogonadotropic hypogonadism in human and mice. Aim. Our aim was to study regulation of the KiSS-1/GPR54 system using a previously characterized primary culture of human fetal GnRH-secreting neuroblasts, FNC-B4. METHODS: KiSS-1/GPR54 gene and protein expressions in FNC-B4 were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), immunocytochemistry, and Western blot. Expression of kisspeptin and GPR54 in fetal olfactory mucosa (OM), from which FNC-B4 cells were derived, was analyzed with confocal microscopy. MAIN OUTCOME MEASURES: Regulation of KiSS-1/GPR54 expression in FNC-B4 was evaluated in response to sexual steroids and leptin. Effect of kisspeptin on GnRH secretion and migration in FNC-B4 was also investigated. RESULTS: Kisspeptin and GPR54 were immunolocalized and co-expressed with GnRH in OM and FNC-B4 cells. Kisspeptin (1 microM, 24 hours) induced GnRH secretion, but not gene expression, and inhibited migration (IC(50) = 6.28 +/- 3.71 nM) in FNC-B4. The 24-hour exposure to increasing concentrations of 17-beta-estradiol (0.01-1 nM) significantly and dose-dependently decreased, whereas androgens (dihydrotestosterone [DHT], 0.01-1 nM) significantly stimulated KiSS-1/GPR54 mRNA. Testosterone (1 nM) showed a stimulatory effect only after blocking its aromatization with letrozole. In addition, leptin (1 nM, 24 hours), an adipocyte-derived hormone acting on the reproductive axis, significantly increased KiSS-1/GPR54 expression in FNC-B4. Immunocytochemistry and Western blot analysis confirmed the regulatory effects found with qRT-PCR. Interestingly, leptin (1 nM, 24 hours) also significantly increased both leptin receptor (LEPR) and androgen receptor (AR) mRNA. DHT (0.01-1 nM) also up-regulated LEPR and AR genes, suggesting a synergistic action between leptin and androgens aimed to up-regulate the KiSS-1/GPR54 system, which, in contrast, was inhibited by estrogens. CONCLUSION: Our results indicate that an interplay between metabolic and sexual hormones may trigger the KiSS-1/GPR54 signaling to GnRH neurons suggesting new mechanisms which regulate puberty onset.

Atorvastatin but not elocalcitol increases sildenafil responsiveness in spontaneously hypertensive rats by regulating the RhoA/ROCK pathway.

Spontaneously hypertensive rats (SHR) are characterized by impaired erectile function and overactivity of the procontractile RhoA/Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) pathway, as compared with their normotensive counterpart, Wistar-Kyoto rats. By measuring the intracavernous pressure:mean arterial pressure (ICP:MAP) ratio after electrostimulation of the cavernous nerve, we confirmed these findings and showed that responsiveness to sildenafil (25 mg/kg by oral gavage) also is hampered in SHR. A 2-week treatment with atorvastatin (5 and 30 mg/kg) improved the sildenafil-induced ICP:MAP increase and normalized RhoA and ROCK2 overexpression in SHR corpora cavernosa (CC). Conversely, other genes, neuronal nitric oxide synthase (NOS), endothelial NOS, and phosphodiesterase 5, were unaffected. In human fetal smooth muscle cells derived from CC (hfPSMC), atorvastatin inhibited RhoA membrane translocation and ROCK activity, as well as RhoA-dependent biologic functions like cell migration and cell proliferation. Atorvastatin's effect on migration was rescued in a dose-dependent manner by geranylgeranyl pyrophosphate, suggesting the involvement of RhoA geranylgeranylation. In hfPSMC, atorvastatin decreased the expression of RhoA-dependent genes such as ROCK2, desmin, alpha-smooth muscle actin, SM22alpha, and myocardin. In contrast to atorvastatin, elocalcitol, a vitamin D analog that also interferes with RhoA activation in SHR bladder, was unable to restore penile responsiveness to sildenafil. In conclusion, atorvastatin, but not elocalcitol, ameliorates sildenafil-induced penile erections in SHR, likely by interfering with RhoA/ROCK signaling within the penis.

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.

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.

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.

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.

Synthesis, biological activity, and three-dimensional quantitative structure-activity relationship model for a series of benzo[c]quinolizin-3-ones, nonsteroidal inhibitors of human steroid 5alpha-reductase 1.

New 5alpha-reductase 1 (5alphaR-1) inhibitors were designed to complete a consistent set of analogues suitable for a 3D QSAR study. These compounds were synthesized by a modification of the aza-Robinson annulation, further functionalized by Pd-catalyzed cross-coupling processes, and were tested with human 5alphaR-1 expressed in Chinese hamster ovary 1827 cells. It turned out that the potency of the resulting inhibitors was strongly dependent on the type of substitution at the 8 position, with the IC(50) values ranging from 8.1 to 1050 nM. The construction of this homogeneous set of molecules allowed a 3D QSAR study. In particular, comparative molecular field analysis (CoMFA) was used to correlate the potency of the inhibitors with their physicochemical features. Highly accurate evaluations of the atomic point charges were carried out by means of quantum chemical calculations at the DFT/B3LYP level of theory followed by the RESP fitting procedure. It turned out that increasing the reliability of electrostatic parameters greatly affected the statistical results of the QSAR analysis. The 3D QSAR model proposed could be very useful in the further development of 5alphaR-1 inhibitors, which are suitable candidates to be evaluated as drugs in the treatment of 5alphaR-1 related diseases such as acne and alopecia in men and hirsutism in women.

The vitamin D receptor agonist elocalcitol upregulates L-type calcium channel activity in human and rat bladder.

Human bladder contraction mainly depends on Ca2+ influx via L-type voltage-gated Ca2+ channels and on RhoA/Rho kinase contractile signaling, which is upregulated in overactive bladder (OAB). Elocalcitol is a vitamin D receptor agonist inhibiting RhoA/Rho kinase signaling in rat and human bladder. Since in the normal bladder from Sprague-Dawley rats elocalcitol treatment delayed the carbachol-induced contraction without changing maximal responsiveness and increased sensitivity to the L-type Ca2+ channel antagonist isradipine, we investigated whether elocalcitol upregulated L-type Ca2+ channels in human bladder smooth muscle cells (hBCs). In hBCs, elocalcitol induced a rapid increase in intracellular [Ca2+], which was abrogated by the L-type Ca2+ channel antagonist verapamil. Moreover, hBCs exhibited L-type voltage-activated Ca2+ currents (I Ca), which were selectively blocked by isradipine and verapamil and enhanced by the selective L-type agonist BAY K 8644. Addition of elocalcitol (10(-7) M) increased L-type I Ca size and specific conductance by inducing faster activation and inactivation kinetics than control and BAY K 8644, while determining a significant negative shift of the activation and inactivation curves, comparable to BAY K 8644. These effects were strengthened in long-term treated hBCs with elocalcitol (10(-8) M, 48 h), which also showed increased mRNA and protein expression of pore-forming L-type alpha(1C)-subunit. In the bladder from Sprague-Dawley rats, BAY K 8644 induced a dose-dependent increase in tension, which was significantly enhanced by elocalcitol treatment (30 microg.kg(-1).day(-1), 2 wk). In conclusion, elocalcitol upregulated Ca2+ entry through L-type Ca2+ channels in hBCs, thus balancing its inhibitory effect on RhoA/Rho kinase signaling and suggesting its possible efficacy for the modulation of bladder contractile mechanisms.

Essential but differential role for CXCR4 and CXCR7 in the therapeutic homing of human renal progenitor cells.

Recently, we have identified a population of renal progenitor cells in human kidneys showing regenerative potential for injured renal tissue of SCID mice. We demonstrate here that among all known chemokine receptors, human renal progenitor cells exhibit high expression of both stromal-derived factor-1 (SDF-1) receptors, CXCR4 and CXCR7. In SCID mice with acute renal failure (ARF), SDF-1 was strongly up-regulated in resident cells surrounding necrotic areas. In the same mice, intravenously injected renal stem/progenitor cells engrafted into injured renal tissue decreased the severity of ARF and prevented renal fibrosis. These beneficial effects were abolished by blocking either CXCR4 or CXCR7, which dramatically reduced the number of engrafting renal progenitor cells. However, although SDF-1-induced migration of renal progenitor cells was only abolished by an anti-CXCR4 antibody, transendothelial migration required the activity of both CXCR4 and CXCR7, with CXCR7 being essential for renal progenitor cell adhesion to endothelial cells. Moreover, CXCR7 but not CXCR4 was responsible for the SDF-1-induced renal progenitor cell survival. Collectively, these findings suggest that CXCR4 and CXCR7 play an essential, but differential, role in the therapeutic homing of human renal progenitor cells in ARF, with important implications for the development of stem cell-based therapies.

Testosterone regulates RhoA/Rho-kinase signaling in two distinct animal models of chemical diabetes.

INTRODUCTION: The contractile RhoA/Rho-kinase (ROCK) signaling pathway is upregulated in penile tissue in animal models of experimental diabetes and has been proposed to contribute to diabetes-related erectile dysfunction (ED). AIM: To investigate the effect of testosterone (T) on the RhoA/ROCK signaling in diabetes. METHODS: We used two distinct animal models of chemical diabetes (alloxan-induced in the rabbit and streptozotocin-induced in the rat) with or not T supplementation. MAIN OUTCOME MEASURES: The effect of diabetes and T supplementation on RhoA/ROCK signaling was evaluated as responsiveness to the selective ROCK inhibitor Y-27632 either by "in vitro" contractility study (diabetic rabbit) or "in vivo" as erectile response elicited by intracavernous injections (diabetic rats). RhoA/ROCK gene and protein expression were also analyzed. RESULTS: In both models, hypogonadism was observed, characterized by reduced T plasma level and androgen-dependent accessory glands atrophy. Diabetic animals showed a significant increase in responsiveness to increasing concentrations of Y-27632. T substitution (30 mg/kg, weekly) completely prevented hypogonadism and diabetes-induced penile hypersensitivity to Y-27632. To test whether this effect was due to a T-dependent regulation of RhoA/ROCK gene expression, we measured RhoA/ROCK mRNA. Both isoforms of ROCK (ROCK1/ROCK2) were analyzed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in rat penile samples. We found that ROCK1 mRNA was significantly increased (P < 0.05) in penile tissues from diabetic animals and maintained at the control values by T, as also confirmed by semiquantitative RT-PCR in rabbit. Conversely, RhoA and ROCK2 mRNA expression was not influenced either by diabetic condition or by T administration. Accordingly, ROCK1 protein expression, as evaluated by Western blot and immunohistochemistry analysis, was increased in penile samples from diabetic animals and normalized by T. CONCLUSIONS: Our data further support the hypothesis that the overexpression of RhoA/ROCK signaling contributes to diabetes-related ED. Moreover, treating hypogonadism in course of diabetes may maintain erectile function also by normalizing RhoA/ROCK pathway upregulation.

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.

Synthesis and activity of 8-substituted benzo[c]quinolizin-3-ones as dual inhibitors of human 5alpha-reductases 1 and 2.

Some potent dual inhibitors of 5alpha-reductases 1 and 2, based on the benzo[c]quinolizin-3-one structure and with IC(50) values ranging between 93 and 166nM for both isozymes, were found. The presence of the F atom on the ester moiety at the position 8 was crucial. This result can help in the design of other potent, dual inhibitors to be developed as drugs in the treatment of 5alpha-reductase related diseases.

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.

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.