Effects of Reduced Extracellular Sodium Concentrations on Cisplatin Treatment in Human Tumor Cells: The Role of Autophagy.

Hyponatremia is the prevalent electrolyte imbalance in cancer patients, and it is associated with a worse outcome. Notably, emerging clinical evidence suggests that hyponatremia adversely influences the response to anticancer treatments. Therefore, this study aims to investigate how reduced extracellular [Na+] affects the responsiveness of different cancer cell lines (from human colon adenocarcinoma, neuroblastoma, and small cell lung cancer) to cisplatin and the underlying potential mechanisms. Cisplatin dose-response curves revealed higher IC50 in low [Na+] than normal [Na+]. Accordingly, cisplatin treatment was less effective in counteracting the proliferation and migration of tumor cells when cultured in low [Na+], as demonstrated by colony formation and invasion assays. In addition, the expression analysis of proteins involved in autophagosome-lysosome formation and the visualization of lysosomal areas by electron microscopy revealed that one of the main mechanisms involved in chemoresistance to cisplatin is the promotion of autophagy. In conclusion, our data first demonstrate that the antitumoral effect of cisplatin is markedly reduced in low [Na+] and that autophagy is an important mechanism of drug escape. This study indicates the role of hyponatremia in cisplatin chemoresistance and reinforces the recommendation to correct this electrolyte alteration in cancer patients.

The Yin and Yang Effect of the Apelinergic System in Oxidative Stress.

Apelin is an endogenous ligand for the G protein-coupled receptor APJ and has multiple biological activities in human tissues and organs, including the heart, blood vessels, adipose tissue, central nervous system, lungs, kidneys, and liver. This article reviews the crucial role of apelin in regulating oxidative stress-related processes by promoting prooxidant or antioxidant mechanisms. Following the binding of APJ to different active apelin isoforms and the interaction with several G proteins according to cell types, the apelin/APJ system is able to modulate different intracellular signaling pathways and biological functions, such as vascular tone, platelet aggregation and leukocytes adhesion, myocardial activity, ischemia/reperfusion injury, insulin resistance, inflammation, and cell proliferation and invasion. As a consequence of these multifaceted properties, the role of the apelinergic axis in the pathogenesis of degenerative and proliferative conditions (e.g., Alzheimer's and Parkinson's diseases, osteoporosis, and cancer) is currently investigated. In this view, the dual effect of the apelin/APJ system in the regulation of oxidative stress needs to be more extensively clarified, in order to identify new potential strategies and tools able to selectively modulate this axis according to the tissue-specific profile.

Hyponatremia Promotes Cancer Growth in a Murine Xenograft Model of Neuroblastoma.

In cancer patients, hyponatremia is detected in about 40% of cases at hospital admission and has been associated to a worse outcome. We have previously observed that cancer cells from different tissues show a significantly increased proliferation rate and invasion potential, when cultured in low extracellular [Na+]. We have recently developed an animal model of hyponatremia using Foxn1nu/nu mice. The aim of the present study was to compare tumor growth and invasivity of the neuroblastoma cell line SK-N-AS in hyponatremic vs. normonatremic mice. Animals were subcutaneously implanted with luciferase-expressing SK-N-AS cells. When masses reached about 100 mm3, hyponatremia was induced in a subgroup of animals via desmopressin infusion. Tumor masses were significantly greater in hyponatremic mice, starting from day 14 and until the day of sacrifice (day 28). Immunohistochemical analysis showed a more intense vascularization and higher levels of expression of the proliferating cell nuclear antigen, chromogranin A and heme oxigenase-1 gene in hyponatremic mice. Finally, metalloproteases were also more abundantly expressed in hyponatremic animals compared to control ones. To our knowledge, this is the first demonstration in an experimental animal model that hyponatremia is associated to increased cancer growth by activating molecular mechanisms that promote proliferation, angiogenesis and invasivity.

Hyponatraemia alters the biophysical properties of neuronal cells independently of osmolarity: a study on Ni(2+) -sensitive current involvement.

What is the central question of this study? Hyponatraemia, an electrolyte disorder encountered in hospitalized patients, can cause neurological symptoms usually attributed to a reduction in plasma osmolarity. Here, we investigated whether low [Na(+) ] per se can cause neuronal changes independent of osmolarity, focusing on involvement of the Na(+) -Ca(2+) exchanger. What is the main finding and its importance? We show that hyponatraemia per se causes alterations of neuronal properties. The novel finding of Na(+) -Ca(2+) exchanger involvement helps us to elucidate the volume regulation following hyponatraemia. This might have relevance in a translational perspective because Na(+) -Ca(2+) exchanger could be a target for novel therapies. Hyponatraemia is the most frequent electrolyte disorder encountered in hospitalized patients, and it can cause a wide variety of neurological symptoms. Most of the negative effects of this condition on neuronal cells are attributed to cell swelling because of the reduction of plasma osmolarity, although in hyponatraemia different membrane proteins are supposed to be involved in the conservation of neuronal volume. We have recently reported detrimental effects of hyponatraemia on two different neuronal cell lines, SK-N-AS and SH-SY5Y, independent of osmotic alterations. In this study we investigated, in the same cell lines, whether hyponatraemic conditions per se can cause electrophysiological alterations and whether these effects vary over time. Accordingly, we carried out experiments in low-sodium medium in either hyposmotic [Osm(-)] or isosmotic [Osm(+)] conditions, for a short (24 h) or long time (7 days). Using a patch pipette in voltage-clamp conditions, we recorded possible modifications of cell capacitance (Cm ) and membrane conductance (Gm ). Our results indicate that in both Osm(-) and Osm(+) medium, Cm and Gm show a similar increase, but such effects are dependent on the time in culture in different ways. Notably, regarding the possible mechanisms involved in the maintenance of Cm , Gm and Gm /Cm in Osm(+) conditions, we observed a greater contribution of the Na(+) -Ca(2+) exchanger compared with Osm(-) and control conditions. Overall, these novel electrophysiological results help us to understand the mechanisms of volume regulation after ionic perturbation. Our results might also have relevance in a translational perspective because the Na(+) -Ca(2+) exchanger can be considered a target for planning novel therapies.

Hyponatremia and Cancer: From Bedside to Benchside.

Hyponatremia is the most common electrolyte disorder encountered in hospitalized patients. This applies also to cancer patients. Multiple causes can lead to hyponatremia, but most frequently this electrolyte disorder is due to the syndrome of inappropriate antidiuresis. In cancer patients, this syndrome is mostly secondary to ectopic secretion of arginine vasopressin by tumoral cells. In addition, several chemotherapeutic drugs induce the release of arginine vasopressin by the hypothalamus. There is evidence that hyponatremia is associated to a more negative outcome in several pathologies, including cancer. Many studies have demonstrated that in different cancer types, both progression-free survival and overall survival are negatively affected by hyponatremia, whereas the correction of serum [Na+] has a positive effect on patient outcome. In vitro studies have shown that cells grown in low [Na+] have a greater proliferation rate and motility, due to a dysregulation in intracellular signalling pathways. Noteworthy, vasopressin receptors antagonists, which were approved more than a decade ago for the treatment of euvolemic and hypervolemic hyponatremia, have shown unexpected antiproliferative effects. Because of this property, vaptans were also approved for the treatment of polycystic kidney disease. In vitro evidence indicated that this family of drugs effectively counteracts proliferation and invasivity of cancer cells, thus possibly opening a new scenario among the pharmacological strategies to treat cancer.

Human benign prostatic hyperplasia stromal cells as inducers and targets of chronic immuno-mediated inflammation.

Benign prostatic hyperplasia (BPH), a highly prevalent prostatic condition, could involve an inflammatory component in disease pathogenesis. In this study, we show that human stromal prostate cells obtained from BPH tissue can actively contribute to the inflammatory process by secreting proinflammatory cytokines as well as chemokines able to recruit lymphomonuclear cells and by acting as APCs. BPH cells express all of the TLRs and their ligation leads to the secretion of CXCL8/IL-8, CXCL10, and IL-6. In addition, BPH cells express costimulatory as well as class I and class II MHC molecules, which activate alloreactive CD4(+) cells that in turn markedly up-regulate IL-12/IL-23p40 and IL-12p75 secretion by BPH cells. Alloreactive CD4(+) cells activated by BPH cells secrete IFN-gamma and IL-17. These cytokines up-regulate IL-6, IL-8, and CXCL10 production by BPH cells, creating a positive feedback loop that can amplify inflammation. IL-8 induces autocrine/paracrine proliferation of BPH cells, indicating also a growth-promoting activity of this chemokine in disease pathogenesis. These results show that human BPH cells represent nonprofessional APCs able to induce and sustain chronic inflammatory processes, supporting the relevance of inflammation in BPH pathogenesis.

Hyponatremia and Oxidative Stress.

Hyponatremia, i.e., the presence of a serum sodium concentration ([Na+]) < 136 mEq/L, is the most frequent electrolyte imbalance in the elderly and in hospitalized patients. Symptoms of acute hyponatremia, whose main target is the central nervous system, are explained by the "osmotic theory" and the neuronal swelling secondary to decreased extracellular osmolality, which determines cerebral oedema. Following the description of neurological and systemic manifestations even in mild and chronic hyponatremia, in the last decade reduced extracellular [Na+] was associated with detrimental effects on cellular homeostasis independently of hypoosmolality. Most of these alterations appeared to be elicited by oxidative stress. In this review, we focus on the role of oxidative stress on both osmolality-dependent and -independent impairment of cell and tissue functions observed in hyponatremic conditions. Furthermore, basic and clinical research suggested that oxidative stress appears to be a common denominator of the degenerative processes related to aging, cancer progression, and hyponatremia. Of note, low [Na+] is able to exacerbate multiple manifestations of senescence and to decrease progression-free and overall survival in oncologic patients.

Low sodium and tolvaptan have opposite effects in human small cell lung cancer cells.

PURPOSE: Hyponatraemia is frequently observed in cancer patients and can be due to the syndrome of inappropriate anti-diuresis (SIAD), related to ectopic vasopressin secretion, particularly in small cell lung cancer (SCLC). Hyponatraemia is associated with a worse outcome in cancer patients. The vasopressin receptor antagonist tolvaptan effectively corrects hyponatraemia secondary to SIAD and there is in vitro evidence that it has also an antiproliferative effect in cancer cells. The purpose of this study was i) to analyse the effect of low serum sodium concentrations ([Na+]) in SCLC cells and ii) to determine whether tolvaptan counteracts tumor progression. METHODS: We evaluated cell proliferation, cell cycle, apoptosis, oxidative stress, invasivity in low [Na+] as well as after exposure to tolvaptan. We also analysed the intracellular signalling pathways involved. RESULTS: In reduced [Na+] cell proliferation was significantly increased compared to normal [Na+] and cells were mostly distributed in the G2/M phase. Apoptosis appeared reduced. In addition, the ability to cross matrigel-coated membranes markedly increased. As observed in other cancer cell models, the expression of the heme-oxigenase-1 gene was increased. Finally, we found that in cells cultured in low [Na+] the RhoA/ROCK1/2 pathway, which is involved in the regulation of actin cytoskeleton, was activated. On the other hand, we found that tolvaptan effectively inhibited cell proliferation, anchorage-independent growth, invasivity and promoted apoptosis. Accordingly, the RhoA/ROCK-1/2 pathway was inhibited. CONCLUSIONS: These findings demonstrate for the first time that low [Na+] favours tumor progression in SCLC cells, whereas tolvaptan effectively inhibits cell proliferation, survival and invasivity.

Serum sodium alterations in SARS CoV-2 (COVID-19) infection: impact on patient outcome.

OBJECTIVE: Hyponatremia is the most common electrolyte disorder in hospitalized patients and occurs in about 30% of patients with pneumonia. Hyponatremia has been associated with a worse outcome in several pathologic conditions The main objective of this study was to determine whether serum sodium alterations may be independent predictors of the outcome of hospitalized COVID-19 patients. DESIGN AND METHODS: In this observational study, data from 441 laboratory-confirmed COVID-19 patients admitted to a University Hospital were collected. After excluding 61 patients (no serum sodium at admission available, saline solution infusion before sodium assessment, transfer from another hospital), data from 380 patients were analyzed. RESULTS: 274 (72.1%) patients had normonatremia at admission, 87 (22.9%) patients had hyponatremia and 19 (5%) patients had hypernatremia. We found an inverse correlation between serum sodium and IL-6, whereas a direct correlation between serum sodium and PaO2/FiO2 ratio was observed. Patients with hyponatremia had a higher prevalence of non-invasive ventilation and ICU transfer than those with normonatremia or hypernatremia. Hyponatremia was an independent predictor of in-hospital mortality (2.7-fold increase vs normonatremia) and each mEq/L of serum sodium reduction was associated with a 14.4% increased risk of death. CONCLUSIONS: These results suggest that serum sodium at admission may be considered as an early prognostic marker of disease severity in hospitalized COVID-19 patients.

Acute vardenafil administration improves bladder oxygenation in spontaneously hypertensive rats.

INTRODUCTION: In human bladder, phosphodiesterase type 5 (PDE5) is present not only in the muscular wall but also in the vascular beds, suggesting a role for PDE5 inhibitors in favoring bladder blood flow and tissue oxygenation. AIM: To investigate whether acute administration of vardenafil could affect bladder oxygenation in spontaneously hypertensive rats (SHR), an animal model of naturally occurring overactive bladder. MAIN OUTCOME MEASURES: The effect of vardenafil on hypoxia-induced alterations was studied in vivo in SHR by acute dosing (10 mg/kg, 90 minutes before sacrifice) and in vitro in human bladder smooth muscle cells (hBCs). METHODS: Bladder oxygenation was detected using the hypoxyprobe immunostaining. The expression of some hypoxia markers (vascular endothelial growth factor [VEGF] and endothelin-1 type B [ETB] receptor) was also evaluated by immunohistochemistry and Western blot. Gene expression in hBC was quantified by real-time reverse transcription-polymerase chain reaction. RESULTS: Rat bladder PDE5 immunopositivity was detected in the muscular wall and in the endothelial and smooth muscle cells of blood vessels. In SHR bladder, a significant increase of hypoxic cells, VEGF, and ETB expression was observed when compared with their normotensive counterpart Wistar Kyoto rats (WKY). Vardenafil treatment dramatically decreased hypoxyprobe staining, as well as VEGF and ETB expression in SHR bladder up to WKY level. Accordingly, in SHR bladder, vardenafil administration significantly blunted relaxation induced by the selective ETB agonist IRL-1620. In hBCs, experimental hypoxia significantly induced gene expression of hypoxia markers (carbonic anhydrase IX and VEGF), which was not changed by simultaneous treatment with vardenafil. Conversely, the hypoxia-related induction of smooth muscle-specific genes (alphaSMA, SM22alpha, and desmin) was significantly reduced by vardenafil. CONCLUSIONS: SHR showed bladder hypoxia which was significantly reduced by acute vardenafil treatment. Thus, besides relaxing muscular wall, PDE5 inhibition may positively affect urinary vesicle blood perfusion.

Sex steroid receptors in male human bladder: expression and biological function.

INTRODUCTION: In male, lower urinary tract symptoms (LUTS) have been associated, beside benign prostatic hyperplasia, to some unexpected comorbidities (hypogonadism, obesity, metabolic syndrome), which are essentially characterized by an unbalance between circulating androgens/estrogens. Within the bladder, LUTS are linked to RhoA/Rho-kinase (ROCK) pathway overactivity. AIM: To investigate the effects of changing sex steroids on bladder smooth muscle. METHODS: ER α, ER ß, GPR30/GPER1 and aromatase mRNA expression was analyzed in male genitourinary tract tissues, and cells isolated from bladder, prostate, and urethra. Estrogen and G1 effect on RhoA/ROCK signaling output like cell migration, gene expression, and cytoskeletal remodeling, and [Ca(2+) ](i) was also studied in hB cells. Contractile studies on bladder strips from castrated male rats supplemented with estradiol and testosterone was also performed. MAIN OUTCOME MEASURES: The effects of classical (ER α, ER ß) and nonclassical (GPR30/GPER1) estrogen receptor ligands (17 ß-estradiol and G1, respectively) and androgens on RhoA/ROCK-.mediated cell functions were studied in hB cells. Contractility studies were also performed in bladder strips from castrated male rats supplemented with testosterone or estradiol. RESULTS: Aromatase and sex steroid receptors, including GPR30, were expressed in human bladder and mediates several biological functions. Both 17 ß-estradiol and G1 activated calcium transients and induced RhoA/ROCK signaling (cell migration, cytoskeleton remodeling and smooth muscle gene expression). RhoA/ROCK inhibitors blunted these effects. Estrogen-, but not androgen-supplementation to castrated rats increased sensitivity to the ROCK inhibitor, Y-27632 in isolated bladder strips. In hB cells, testosterone elicited effects similar to estrogen, which were abrogated by blocking its aromatization through letrozole. CONCLUSION: Our data indicate for the first time that estrogen-more than androgen-receptors up-regulate RhoA/ROCK signaling. Since an altered estrogen/androgen ratio characterizes conditions, such as aging, obesity and metabolic syndrome, often associated to LUTS, we speculate that a relative hyperestrogenism may induce bladder overactivity through the up-regulation of RhoA/ROCK pathway.

Characterization of phosphodiesterase type 5 expression and functional activity in the human male lower urinary tract.

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.

Effects of low extracellular sodium on proliferation and invasive activity of cancer cells in vitro.

PURPOSE: Hyponatremia is the most common electrolyte disorder in hospitalized patients, and its etiopathogenesis is related to an underlying tumor in 14% of cases. Hyponatremia has been associated with a worse outcome in several pathologies, including cancer, in which the leading cause of this electrolyte alteration is the syndrome of inappropriate antidiuresis. The aim of this study was to analyze in vitro the effects of low extracellular [Na+] in cancer progression. MATERIALS AND METHODS: We used a previously validated experimental model of chronic hyponatremia to characterize the effects of low extracellular [Na+] in different human cancer cell lines: pancreatic adenocarcinoma (PANC-1), neuroblastoma (SK-N-AS, SH-SY5Y), colorectal adenocarcinoma (HCT-8), chronic myeloid leukemia (K562). RESULTS: Our results demonstrate a direct relationship between low [Na+], reduced cell adhesion and increased invasion and proliferation in all cell lines tested. Accordingly, the number of tumor colonies grown in soft agar and the expression of collagenases type IV (metalloproteinases 2 and 9) were markedly higher in cancer cells exposed to reduced extracellular [Na+]. Gene analysis showed an upregulation of molecular pathways involved in oxidative stress (heme oxygenase 1) and in proliferation and invasion (RhoA, ROCK-1, ROCK-2). The activation of RhoA/ROCK pathway was paralleled by a deregulation of the cytoskeleton-associated proteins, resulting in the promotion of actin cytoskeletal remodeling and cell invasion. CONCLUSIONS: Overall, our data demonstrate for the first time that low [Na+] promotes cancer progression in vitro, thus suggesting that hyponatremia is not a simple bystander of disease severity in cancer.

The vitamin D receptor agonist elocalcitol inhibits IL-8-dependent benign prostatic hyperplasia stromal cell proliferation and inflammatory response by targeting the RhoA/Rho kinase and NF-kappaB pathways.

BACKGROUND: Benign prostatic hyperplasia (BPH) is characterized by an important inflammatory component. Stimulation of human prostate stromal cells from BPH tissues with proinflammatory cytokines leads to secretion of IL-8, a chemokine involved in BPH pathogenesis. The vitamin D receptor (VDR) agonist elocalcitol can arrest prostate growth in BPH patients, but its mechanism of action in this pathology is still incompletely understood. METHODS: IL-8 levels were measured by real-time RT-PCR and ELISA. NF-kappaB translocation and COX-2 expression were evaluated by confocal microscopy. RhoA and Rho-kinase (ROCK) gene expression and functional activity were studied by real-time RT-PCR, immuno-kinase assays, Western blot analysis, confocal microscopy, and cell invasion. RESULTS: Stimulation of BPH cells with IL-8 activates the calcium-sensitizing RhoA/ROCK pathway, as demonstrated by the increased membrane translocation of RhoA and by phosphorylation of the ROCK substrate myosin phosphatase target subunit 1 (MYPT-1). In agreement with these data, C3 exoenzyme, a selective RhoA inhibitor, inhibits IL-8-induced invasion of BPH cells. The VDR agonist elocalcitol significantly inhibits IL-8 production by BPH cells stimulated with inflammatory cytokines, and IL-8-induced proliferation of BPH cells. In addition, elocalcitol inhibits IL-8-induced membrane translocation of RhoA and MYPT-1 phosphorylation in BPH cells, and inhibits dose-dependently their IL-8-dependent invasion. The inhibition induced by elocalcitol of IL-8 production by BPH cells is accompanied by decreased COX-2 expression and PGE(2) production and by arrest of NF-kappaB p65 nuclear translocation, associated with inhibition of the RhoA/ROCK pathway. CONCLUSIONS: These data provide a mechanistic explanation for the anti-proliferative and anti-inflammatory properties of elocalcitol in BPH cells.

Dihydrotestosterone and leptin regulate gonadotropin-releasing hormone (GnRH) expression and secretion in human GnRH-secreting neuroblasts.

INTRODUCTION: The reversal of hypogonadotropic hypogonadism (HH), occurring after discontinuation of testosterone therapy in adolescents with delayed puberty and in a small percentage of adults with congenital HH, suggests a role for androgens in favoring a spontaneous recovery of reproductive function. AIM: We investigated the effect of androgens and leptin on gonadotropin-releasing hormone (GnRH) expression and secretion in human GnRH-secreting neuroblasts (FNC-B4). METHODS: Quantitative real-time polymerase chain reaction RT-PCR for mRNA expression and radioimmunoassay for GnRH secretion were used. Immunohistochemical studies assessed GnRH protein expression. FNC-B4 migration was analyzed with multiwell Boyden chamber technique. MAIN OUTCOME MEASURES: Effects of the non-aromatizable androgen dihydrotestosterone (DHT) and leptin in FNC-B4 were tested after 24 and 48 hours. RESULTS: Exposure to increasing concentrations of DHT after 24 hours significantly stimulated GnRH mRNA in FNC-B4. This effect was still present after prolonged exposure (48 hours). Similarly, treatment with leptin significantly induced GnRH mRNA after 24 hours, but not at 48 hours. Interestingly, mRNA for leptin receptors (LEPR) was significantly reduced after 48 hours of leptin, while, at this time point, it was stimulated by DHT. Coincubation for 48 hours with leptin and DHT maintained the stimulatory effect on both GnRH and LEPR mRNA, suggesting that DHT could stabilize the leptin effect by preventing downregulation of LEPR. Similar results were obtained for GnRH protein expression analysis. Moreover, both DHT and leptin increased GnRH release into the culture medium. We also found that DHT or leptin treatment significantly increased FNC-B4 basal migration. As we previously found that GnRH stimulates FNC-B4 migration, we hypothesized that this effect could be mediated by DHT- and leptin-induced GnRH release. Accordingly, the GnRH antagonist cetrorelix inhibited DHT- and leptin-induced migration. CONCLUSION: Our results suggest that androgens (adequate hormonal status) could have a positive effect on GnRH neuronal activity by synergizing with leptin (adequate energy status) in the regulatory mechanisms required for reproductive and sexual fitness.

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.

Atorvastatin ameliorates sildenafil-induced penile erections in experimental diabetes by inhibiting diabetes-induced RhoA/Rho-kinase signaling hyperactivation.

INTRODUCTION: One of the proposed mechanisms responsible for diabetes-related erectile dysfunction (ED) is overactivity of RhoA/ROCK signaling, as seen in experimental models of chemical diabetes. AIM: Because statins may interfere with RhoA/Rho-kinase (ROCK) signaling through the reduction of geranyl-geranyl pyrophosphate (GGPP), required for RhoA activation, we investigated whether atorvastatin ameliorated diabetes-related ED. METHODS: Streptozotocin-induced (8 weeks) diabetic rats and alloxan-induced (8 weeks) diabetic rabbits received atorvastatin (5 mg/kg daily) for the last 2 weeks. In vitro contractility studies were conducted in the rabbit model. In the rat model, sildenafil effect on electrical stimulation (ES)-induced erection was investigated. Atorvastatin action was also analyzed using human fetal penile smooth muscle cells (hfPSMCs) exposed to low (5 mM), high (22 mM), and very high (40 mM) glucose. MAIN OUTCOME MEASURES: Atorvastatin effect on hyperglycemia-induced RhoA/ROCK signaling was evaluated using the ROCK inhibitor Y-27632 in both animal models and by analyzing functional effects downstream to RhoA activation in hfPSMCs. RESULTS: In both diabetic models, atorvastatin did not affect glycemia, lipid plasma levels, and the hypogonadal state. In diabetic rats, atorvastatin ameliorated the erectile response to the ES of the cavernous nerve and normalized sildenafil effect on erectile function, strongly decreased by diabetes. In penile tissue from diabetic animals, atorvastatin completely restored the diabetes-induced hypersensitivity to Y-27632 and prevented RhoA membrane translocation/activation. In hfPSMCs, high glucose significantly increased not only membrane RhoA expression, but also ROCK activity (increased phosphorylation of the ROCK substrate myosin phosphatase target subunit 1) and several RhoA-dependent functions such as proliferation, migration, and smooth muscle-related gene expression. Atorvastatin restored all the high-glucose-induced effects, an action specifically reverted by GGPP. CONCLUSION: Atorvastatin improves diabetes-related ED and restores sildenafil responsiveness, most probably by inhibiting RhoA/ROCK signaling, which underlies several high-glucose-induced derangements in penile smooth muscle cell commitment.

Vardenafil modulates bladder contractility through cGMP-mediated inhibition of RhoA/Rho kinase signaling pathway in spontaneously hypertensive rats.

INTRODUCTION: Phosphodiesterase type 5 inhibitors (PDE5i), the most widely used drugs for erectile dysfunction, could also improve lower urinary tract symptoms, essentially due to overactive bladder (OAB), a condition hypothesized to be a result of an increased RhoA/Rho-kinase (ROCK) signaling. Phosphorylation/inactivation of RhoA by cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) activity has been described in vascular smooth muscle. AIM: The aim of this paper was to investigate whether vardenafil-induced cGMP accumulation reduces RhoA/ROCK signaling in bladder. METHODS: Spontaneously hypertensive rats (SHRs), a strain genetically prone to develop OAB, were treated with vardenafil (10 mg/kg/day) for 2 weeks. Wistar-Kyoto rats (WKY) were used as control. In vitro experiments were performed in human bladder smooth muscle cells (hBCs). MAIN OUTCOME MEASURES: Urodynamic parameters were registered in vivo in anesthetized WKY and SHRs. RhoA/ROCK activity in bladder was evaluated by molecular and functional studies in tissues and cells. RESULTS: The intercontraction interval and bladder capacity, and were decreased in SHRs and restored by vardenafil. The in vitro relaxant effect of the ROCK inhibitor Y-27632 was higher in bladder strips from SHR than from WKY and reduced by vardenafil. Nomega-nitro-L-arginine-methyl-ester (a NO-synthase inhibitor, 40 mg/kg/day during the last week of the 2-week treatment with vardenafil) partially antagonized vardenafil effect on Y-27632 responsiveness. Vardenafil prevented RhoA membrane translocation/activation, decreased ROCK activity, and increased cGMP levels in vivo (rat) and in vitro (hBCs). Exposing hBCs to vardenafil increased Ser(188) RhoA phosphorylation, to the same extent as the PDE5-insensitive PKG agonist Sp-8-Br-PET-cGMP. Moreover, vardenafil inhibited several RhoA-dependent functions in hBCs, including smooth muscle gene transcription and endothelin-1-induced migration. These effects were reverted by the PKG inhibitor KT 5823, further suggesting a cGMP/PKG-dependency. In hBCs, vardenafil was active in the low nanomolar range. CONCLUSIONS: This is the first study demonstrating that the effect of vardenafil on OAB could be partially determined by a cGMP-dependent RhoA/ROCK signaling inhibition.

Estrogens regulate humans and rabbit epididymal contractility through the RhoA/Rho-kinase pathway.

INTRODUCTION: We have previously demonstrated that oxytocin (OT) and endothelin-1 (ET-1) peripherally regulate epididymal motility in an estrogen-dependent way. Because RhoA/Rho-kinase (ROCK) pathway is a contractile effector downstream to both OT and ET-1 receptors, we hypothesized an estrogenic modulation of OT- and ET-1-induced contraction through the up-regulation of RhoA/ROCK signaling. AIM: To evaluate the effect of changing endocrine milieu on RhoA/ROCK pathway in the epididymis. METHODS: We induced a pharmacological hypogonadotropic hypogonadism in rabbits and replaced hypogonadal animals with different sex steroids (testosterone, T, or estradiol valerate, [E(2v)]). Effects of estrogen deprivation were also evaluated in rabbits chronically treated with the P450-aromatase inhibitor letrozole. An "in vitro" model of human epididymal smooth muscle cells was established and stimulated with sex hormones (72 hours). Protein and mRNA expression and functional activity of RhoA/ROCK signaling were studied by quantitative reverse transcriptase-polymerase chain reaction, immunohistochemistry, western blot analysis, cell migration and by "in vitro" contractility studies using the ROCK inhibitor Y-27632. MAIN OUTCOME MEASURES: Effects of sex steroids on expression and functional activation of RhoA/ROCK signaling in rabbit epididymis and human epididymal smooth muscle cells. RESULTS: The relaxant effect of Y-27632 on ET-1-pre-contracted epididymal strips was significantly reduced in hypogonadal rabbits, as well as in letrozole-treated animals. T supplementation normalized T plasma levels, but not Y-27632 epididymal strip sensitivity. E(2)v not only completely restored Y-27632 responsiveness but even amplified it, indicating an estrogenic up-regulation of RhoA/ROCK pathway. Accordingly, ROCK1 protein and gene expressions were strongly induced by E(2)v but not by T. The estrogen-induced up-regulation of RhoA/ROCK signaling was confirmed in human epididymal smooth muscle cells. CONCLUSIONS: Our results suggest that estrogens regulate epididymal motility by increasing RhoA/ROCK signaling, and therefore calcium sensitivity, which tunes up responsiveness to contractile factors.

Testosterone partially ameliorates metabolic profile and erectile responsiveness to PDE5 inhibitors in an animal model of male metabolic syndrome.

INTRODUCTION: Metabolic syndrome (MetS) is a clustering of cardio-metabolic risk factors (hyperglycemia, hypertension, dyslipidemia, visceral fat accumulation) that is also associated with hypogonadism and erectile dysfunction (ED). AIM: To clarify the relationships among MetS, hypogonadism, and ED, we developed an animal model of MetS. METHODS: Male rabbits fed a high-fat diet (HFD), with or without testosterone (T) supplementation, were compared with control rabbits (fed a standard chow) and with rabbits made hypogonadal by a single injection of a long-acting GnRH-analog, triptorelin. MAIN OUTCOME MEASURES: Evaluation of metabolic disturbances (plasma glucose, cholesterol, triglycerides, testosterone, LH, FSH level, glucose tolerance, mean arterial pressure, visceral fat accumulation), and corpora cavernosa (CC) relaxant capacity (in vitro contractility study) in HFD animals as compared with control, GnRH analog-treated rabbits, and T-supplemented HFD rabbits. RESULTS: HFD rabbits showed all the features of MetS. HFD induced hypogonadotropic hypogonadism is characterized by a reduction of plasma T, FSH, LH levels, testis and seminal vesicles weight, and testicular steroidogenic enzymes. Such a phenotype is similar to that induced by triptorelin administration. A reduced GnRH immunopositivity in hypothalamus suggests a central origin of HFD-related hypogonadism. HFD also induced penile alterations, as demonstrated by a reduction of acetylcholine-and electrical field stimulation-induced CC relaxation, hyper-responsiveness to the NO donor, SNP, and unresponsiveness to PDE5 inhibitors. Similar penile alterations were observed in triptorelin treated rabbit. In HFD, as well as in triptorelin treated rabbits, PDE5 and eNOS mRNA expression quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) were significantly decreased. T administration prevented almost all penile alterations observed in HFD rabbits. T treatment dramatically reduced HFD-induced visceral obesity, partially ameliorating also the metabolic profile. CONCLUSION: We have developed an animal model of MetS associated with hypogonadotropic hypogonadism and penile alterations including unresponsiveness to PDE5 inhibitors. T supplementation was able to partially revert HFD-induced phenotype.