A LC-MS/MS method for the simultaneous determination of 6-cyanodopamine, 6-nitrodopamine, 6-nitrodopa, 6-nitroadrenaline and 6-bromodopamine in human plasma and its clinical application in patients with chronic kidney disease.

The aim of this study was to develop a high-performance liquid chromatography-tandem mass spectrometry method for the determination of 6-cyanodopamine, 6-nitrodopamine, 6-nitrodopa, 6-nitroadrenaline and 6-bromodopamine in human plasma samples. Strata-X 33 µm solid-phase extraction cartridges were used for the extraction of the catecholamines from human plasma samples. The catecholamines were separated in a 150 × 3 mm Shim-pack GIST C18-AQ column with 3 µm particle size, placed in an oven at 40°C and perfused with 82% mobile phase A (acetonitrile-H2O; 90:10, v/v) + 0.4% acetic acid and 18% mobile phase B (deionized H2O) + 0.2% formic acid at a flow rate of 340 µl/min in isocratic mode. The injected volume was 4 µl and the run lasted 4 min. The method was linear from 0.1 to 20 ng/ml and the lower limit of quantification was 0.1 ng/ml for all analytes. The method was applied to evaluate the plasma levels of catecholamines in plasma of patients with chronic kidney disease and allowed the detection for the first time of circulating levels of the novel catecholamines 6-bromodopamine and 6-cyanodopamine.

Periprostatic adipose tissue (PPAT) supernatant from obese mice releases anticontractile substances and increases human prostate epithelial cell proliferation: the role of nitric oxide and adenosine.

Background: The prostate gland is surrounded by periprostatic adipose tissue (PPAT) that can release mediators that interfere in prostate function. In this study, we examined the effect of periprostatic adipose tissue supernatant obtained from obese mice on prostate reactivity in vitro and on the viability of human prostatic epithelial cell lines. Methods: Male C57BL/6 mice were fed a standard or high-fat diet after which PPAT was isolated, incubated in Krebs-Henseleit solution for 30 min (without prostate) or 60 min (with prostate), and the supernatant was then collected and screened for biological activity. Total nitrate and nitrite (NOx-) and adenosine were quantified, and the supernatant was then collected and screened for biological activity. NOx- and adenosine were quantified. Concentration-response curves to phenylephrine (PE) were obtained in prostatic tissue from lean and obese mice incubated with or without periprostatic adipose tissue. In some experiments, periprostatic adipose tissue was co-incubated with inhibitors of the nitric oxide (NO)-cyclic guanosine monophosphate pathway (L-NAME, 1400W, ODQ), adenylate cyclase (SQ22536) or with adenosine A2A (ZM241385), and A2B (MRS1754) receptor antagonists. PNT1-A (normal) and BPH-1 (hyperplasic) human epithelial cells were cultured and incubated with supernatant from periprostatic adipose tissue for 24, 48, or 72 h in the absence or presence of these inhibitors/antagonists, after which cell viability and proliferation were assessed. Results: The levels of NOx- and adenosine were significantly higher in the periprostatic adipose tissue supernatant (30 min, without prostate) when compared to the vehicle. A trend toward an increase in the levels of NOX was observed after 60 min. PPAT supernatant from obese mice significantly reduced the PE-induced contractions only in prostate from obese mice. The co-incubation of periprostatic adipose tissue with L-NAME, 1400W, ODQ, or ZM241385 attenuated the anticontractile activity of the periprostatic adipose tissue supernatant. Incubation with the supernatant of periprostatic adipose tissue from obese mice significantly increased the viability of PNT1-A cells and attenuated expression of the apoptosis marker protein caspase-3 when compared to cells incubated with periprostatic adipose tissue from lean mice. Hyperplastic cells (BPH-1) incubated with periprostatic adipose tissue from obese mice showed greater proliferation after 24 h, 48 h, and 72 h compared to cells incubated with culture medium alone. BPH-1 cell proliferation in the presence of PPAT supernatant was attenuated by NO-signaling pathway inhibitors and by adenosine receptor antagonists after 72 h. Conclusion: NO and adenosine are involved in the anticontractile and pro-proliferative activities of periprostatic adipose tissue supernatant from obese mice. More studies are needed to determine whether the blockade of NO and/or adenosine derived from periprostatic adipose tissue can improve prostate function.

Adsorption kinetics of ciprofloxacin and ofloxacin by green-modified carbon nanotubes.

This paper investigated the uptake of CIP and OFL in single and multicomponent adsorptive systems using modified carbon nanotubes (CNTs) as adsorbent material. The characterization analyses of the pre- and post-process material by XPS, TG/DTG, FT-IR, SEM/EDS, and XRD helped in the elucidation of the mechanisms, indicating greater involvement of n-n and π -π interactions. In the kinetic studies, the simple systems with CIP and OFL were similar, both showed equilibrium time around 20/30 min and increased adsorptive capacity with increasing initial drug concentration. In the multicomponent system, different fractions of CIP and OFL were tested and the time to reach equilibrium also varied between 20 and 30 min. In general, the adsorption capacity of CIP is slightly lower than that of OFL under the conditions tested. The selectivity analysis of the system showed that the selectivity's of the two drugs are identical in equimolar fractions. The mathematical modeling of the kinetic data indicated that in monocomponent systems, the model of pseudo-second order (PSO) adequately described both CIP and OFL kinetics. Furthermore, with the implementation of Artificial Neural Networks (ANN), it was possible to obtain a more assertive prediction of the behavior of single and binary systems.

Enhanced RAGE Expression and Excess Reactive-Oxygen Species Production Mediates Rho Kinase-Dependent Detrusor Overactivity After Methylglyoxal Exposure.

Methylglyoxal (MGO) is a highly reactive dicarbonyl compound implicated in diabetes-associated diseases. In vascular tissues, MGO induces the formation of advanced glycation end products (AGEs) that bounds its receptor RAGE, initiating the downstream tissue injury. Outside the cardiovascular system, MGO intake produces mouse voiding dysfunction and bladder overactivity. We have sought that MGO-induced bladder overactivity is due to activation of AGE-RAGE-reactive-oxygen species (ROS) signaling cascade, leading to Rho kinase activation. Therefore, female mice received 0.5% MGO orally for 12 weeks, after which in vitro bladder contractions were evaluated in the presence or not of superoxide dismutase (PEG-SOD) or the Rho kinase inhibitor Y27632. Treatment with MGO significantly elevated the serum levels of MGO and fluorescent AGEs, as well as the RAGE immunostaining in the urothelium, detrusor, and vascular endothelium. RAGE mRNA expression in the bladder was also higher in the MGO group. Methylglyoxal significantly increased the ROS production in both urothelium and detrusor smooth muscle, with the increases in detrusor markedly higher than urothelium. The bladder activity of superoxide dismutase (SOD) was significantly reduced in the MGO group. Gene expressions of L-type Ca2+ channels, RhoA, ROCK-1, and ROCK-2 in bladder tissues were significantly elevated in the MGO group. Increased bladder contractions to electrical-field stimulation, carbachol α,ß-methylene ATP, and extracellular Ca2+ were observed after MGO exposure, which was significantly reduced by prior incubation with either PEG-SOD or Y27632. Overall, our data indicate serum MGO accumulation elevates the AGEs levels and activates the RAGE-ROS signaling leading to Rho kinase-induced muscle sensitization, ultimately leading to detrusor overactivity.

Reduced blood pressure in sickle cell disease is associated with decreased angiotensin converting enzyme (ACE) activity and is not modulated by ACE inhibition.

BACKGROUND: Sickle cell disease (SCD) incurs vaso-occlusive episodes and organ damage, including nephropathy. Despite displaying characteristics of vascular dysfunction, SCD patients tend to present relatively lower systemic blood pressure (BP), via an unknown mechanism. We investigated associations between BP and renin-angiotensin-system (RAS) components in SCD and determined whether an inhibitor of angiotensin converting enzyme (ACE; often used to slow SCD glomerulopathy) further modulates BP and RAS components in a murine model of SCD. METHODS: BP was compared in human subjects and mice with/without SCD. Plasma angiotensin II, ACE and renin were measured by immunoassay. BP was reevaluated after treating mice with enalapril (25 mg/kg, 5x/week) for 5 weeks; plasma and organs were stored for angiotensin II and ACE activity measurement, and quantitative real-time PCR. RESULTS: Diastolic BP and systolic BP were significantly lower in patients and mice with SCD, respectively, compared to controls. Reduced BP was associated with increased plasma renin and markers of kidney damage (mice) in SCD, as well as significantly decreased plasma ACE concentrations and ACE enzyme activity. As expected, enalapril administration lowered BP, plasma angiotensin II and organ ACE activity in control mice. In contrast, enalapril did not further reduce BP or organ ACE activity in SCD mice; however, plasma angiotensin II and renin levels were found to be significantly higher in enalapril-treated SCD mice than those of treated control mice. CONCLUSION: Relative hypotension was confirmed in a murine model of SCD, in association with decreased ACE concentrations in both human and murine disease. Given that ACE inhibition has an accepted role in decreasing BP, further studies should investigate mechanisms by which ACE depletion, via both Ang II-dependent and alternative pathways, could contribute to reduce BP in SCD and understand how ACE inhibition confers Ang II-independent benefits on kidney function in SCD.

Selective Pharmacological Inhibition of NOX2 by GSK2795039 Improves Bladder Dysfunction in Cyclophosphamide-Induced Cystitis in Mice.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory disease without consistently effective treatment. Among the many mediators implicated in cystitis, the overproduction of reactive oxygen species (ROS) seems to play a key role, although the main source of ROS remains unclear. This study aimed to investigate the contribution of NADPH oxidase (NOX) isoforms in ROS generation and the voiding dysfunction of cyclophosphamide (CYP, 300 mg/Kg, ip, 24 h)-induced cystitis in adult female mice, a well-recognized animal model to study IC/BPS, by using GKT137831 (5 mg/Kg, ip, three times in a 24 h period) or GSK2795039 (5 mg/Kg, ip, three times in a 24 h period) to inhibit NOX1/4 or NOX2, respectively. Our results showed that treatment with GSK2795039 improved the dysfunctional voiding behavior induced by CYP, reduced bladder edema and inflammation, and preserved the urothelial barrier integrity and tight junction occludin expression, besides inhibiting the characteristic vesical pain and bladder superoxide anion generation. In contrast, the NOX1/4 inhibitor GKT137831 had no significant protective effects. Taken together, our in vivo and ex vivo data demonstrate that NOX2 is possibly the main source of ROS observed in cystitis-induced CYP in mice. Therefore, selective inhibition of NOX2 by GSK2795039 may be a promising target for future therapies for IC/BPS.

Methylglyoxal Exacerbates Lipopolysaccharide-Induced Acute Lung Injury via RAGE-Induced ROS Generation: Protective Effects of Metformin.

PURPOSE: Methylglyoxal (MGO) is a highly reactive dicarbonyl species implicated in diabetic-associated diseases. Acute lung injury (ALI) symptoms and prognosis are worsened by diabetes and obesity. Here, we hypothesized that elevated MGO levels aggravate ALI, which can be prevented by metformin. Therefore, this study evaluated the lung inflammation in lipopolysaccharide (LPS)-exposed mice pretreated with MGO. METHODS: C57Bl/6 male mice treated or not with MGO for 12 weeks were intranasally instilled with LPS (30 µg) to induce ALI, and metformin (300 mg/kg) was given as gavage in the last two weeks of treatment. After 6 h, bronchoalveolar lavage fluid (BALF) and lung tissues were collected to quantify the cell infiltration, cytokine levels, reactive-oxygen species (ROS) production, and RAGE expression. RESULTS: LPS exposure markedly increased the neutrophil infiltration in BALF and lung tissue, which was accompanied by higher levels of IFN-γ, TNF-α and IL-1ß compared with untreated group. MGO treatment significantly increased the airways neutrophil infiltration and mRNA expressions of TNF-α and IL-1ß, whereas COX-2 expression remained unchanged. In lung tissues of LPS-exposed mice, MGO treatment significantly increased the immunostaining and mRNA expression of RAGE, and the ROS levels. Serum MGO concentration achieved after 12-week intake was 9.2-fold higher than control mice, which was normalized by metformin treatment. Metformin also reduced the inflammatory markers in response to MGO. CONCLUSION: MGO intake potentiates the LPS-induced ALI, increases RAGE expression and ROS generation, which is normalized by metformin. MGO scavengers may be a good adjuvant therapy to reduce ALI in patients with cardiometabolic diseases.

Metformin abrogates the voiding dysfunction induced by prolonged methylglyoxal intake.

Methylglyoxal (MGO) is a reactive carbonyl species found at high levels in blood of diabetic patients. The anti-hyperglycemic drug metformin can scavenger MGO and reduce the formation of advanced glycation end products (AGEs). Here, we aimed to investigate if MGO-induced bladder dysfunction can be reversed by metformin. Male C57/BL6 mice received 0.5% MGO in drinking water for 12 weeks, and metformin (300 mg/kg, daily gavage) was given in the last two weeks. The bladder functions were evaluated by performing voiding behavior assays, cystometry and in vitro bladder contractions. MGO intake markedly elevated the levels of MGO and fluorescent AGEs in serum and reduced the mRNA expression and activity of glyoxalase (Glo1) in bladder tissues. Glucose levels were unaffected among groups. MGO intake also increased the urothelium thickness and collagen content of the bladder. Void spot assays in conscious mice revealed an increased void volume in MGO group. The cystometric assays in anesthetized mice revealed increases of basal pressure, non-voiding contractions frequency, bladder capacity, inter-micturition pressure and residual volume, which were accompanied by reduced voiding efficiency in MGO group. In vitro bladder contractions to carbachol, α,ß-methylene ATP and electrical-field stimulation were significantly greater in MGO group. Metformin normalized the changes of MGO and AGEs levels, Glo1 expression and activity, urothelium thickness and collagen content. The MGO-induced voiding dysfunction were all restored by metformin treatment. Our findings strongly suggest that the amelioration of MGO-induced voiding dysfunction by metformin relies on its ability to scavenger MGO, preventing its accumulation in blood.

Lipopolysaccharide reduces urethral smooth muscle contractility via cyclooxygenase activation.

Lipopolysaccharide (LPS) is a component of gram-negative bacteria wall that elicits inflammatory response in the host through the toll-like receptor 4 (TLR4) activation. In the lower urinary tract (LUT), bacteria-derived LPS has been associated with lower urinary tract symptoms (LUTS); however, little is known about the effects of LPS in the urethral smooth muscle (USM). In the present study, we evaluated the functional and molecular effects of LPS in mouse USM in vitro, focusing on the LPS-induced TLR4-signaling pathway. Male C57BL6/JUnib and TLR4 knockout mice (TLR4 KO) were used. The USM contraction was performed in the presence of LPS (62.5-500 µg/mL), indomethacin (10 µM), L-NAME (100 µM), and TAK 242 (1 µM). The RT-PCR assay for the IL-1ß, NF-kB, and COX-2 genes was also evaluated in the presence of LPS (125 µg/mL) and caspase 1 inhibitor (20 µM). Our results showed that LPS reduces mouse USM contraction elicited by phenylephrine and vasopressin. This LPS-induced urethral inhibitory effect was not reversed by the TLR4 inhibition or its absence in the TLR4 KO mice. Conversely, indomethacin (but not L-NAME) reversed the LPS-induced USM hypocontractility. Molecular protocols indicated upregulation of IL-1ß, NF-kß, and COX-2 mRNA upon LPS incubation, which were blunted by caspase 1 inhibition. Our data showed that LPS reduced mouse USM contraction independently of TLR4 activation, involving caspase 1 and IL1ß, NF-kB, and COX-2 gene overexpression. Therefore, this alternative pathway might be a valuable target to reduce the LPS-induced urethral dysfunction under infection and inflammatory conditions.

Preserved activity of soluble guanylate cyclase (sGC) in iliac artery from middle-aged rats: Role of sGC modulators.

Vascular aging leads to structural and functional changes. Iliac arteries (IA) provide blood flow to lower urinary tract and pelvic ischemia has been reported as an important factor for bladder remodeling and overactivity. Dysfunction of the nitric oxide (NO)-cyclic guanosine monophosphate pathway (cGMP) is one factor involved in the development of lower urinary tract (LUT) disorders. Therefore, we hypothesized that ageing-associated LUT disorders is a consequence of lower cGMP productions due to an oxidation of soluble guanylate cylase (sGC) that results in local ischemia. In the present study IA from middle-aged and young rats were isolated and the levels of NO, reactive oxygen species (ROS), the gene expression of the enzymes involved in the NO-pathway and concentration-response curves to the soluble guanylate (sGC) stimulator (BAY 41-2272), sGC activator (BAY 58-2667), tadalafil, acetylcholine (ACh) and sodium nitroprusside (SNP) were determined. In IA from middle-aged rats the gene expression for endothelial nitric oxide synthase and the ROS were lower and higher, respectively than the young group. The relaxations induced by ACh and SNP were significantly lower in IA from middle-aged rats. In IA from middle-aged rats the mRNA expression of PDE5 was 55% higher, accompanied by lower relaxation induced by tadalafil. On the other hand, the gene expression for sGCα1 were similar in IA from both groups. Both BAY 41-2272 and BAY 58-2667 produced concentration-dependent relaxations in IA from both groups, however, the latter was 9-times more potent than BAY 41-2272 and produced similar relaxations in IA in both middle-aged and young groups. Yet, the sGC oxidant, ODQ increased the relaxation and the cGMP levels induced by BAY 58-2667. On the other hand, in tissues stimulated with SNP, tadalafil and BAY-2272, the intracellular levels of cGMP were lower in IA from middle-aged than young rats. In conclusion, our results clearly showed that the relaxations induced by the endothelium-dependent and -independent agents, by the PDE5 inhibitor and by sGC stimulator were impaired in IA from aged rats, while that induced by sGC activator was preserved. It suggests that sGC activator may be advantageous in treating ischemia-related functional changes in the lower urinary tract organs in situations where the NO levels are reduced.

Methylglyoxal, a Reactive Glucose Metabolite, Induces Bladder Overactivity in Addition to Inflammation in Mice.

Diabetic bladder dysfunction (DBD) is one of the most common complication of diabetes. Methylglyoxal (MGO), a highly reactive dicarbonyl compound formed as a by-product of glycolysis, is found at high levels in plasma of diabetic patients. Here, we explored the effects of chronic administration of MGO on micturition pattern (cystometry) and bladder contractility in vitro in healthy male C57/BL6 mice. Methylglyoxal was given at 0.5% in drinking water for 4 weeks. Exposure to MGO led to bladder tissue disorganization, edema of lamina propria, partial loss of urothelium and multiple leukocyte infiltrates. Filling cystometry revealed significant increases of micturition frequency and number of non-voiding contractions (NVCs) in the MGO group, clearly indicating an overactive bladder profile. Bladder contractions induced by electrical-field stimulation (EFS) and carbachol were significantly higher in the MGO group, while the muscarinic M2 and M3 mRNA expressions remained unchanged between groups. Additionally, MGO exposure induced upregulation of TRPA1 and down-regulation of TRPV1 and TRPV4 in bladder tissues. Methylglyoxal did not change the mRNA expression of the advanced glycation end products receptor (RAGE), but markedly increased its downstream NF-κB - iNOS signaling. The mRNA expression of cyclooxygenase-2 (COX-2) and reactive-oxygen species (ROS) levels remained unchanged. Altogether, our data show that 4-week MGO intake in mice produces an overactive bladder phenotype in addition to bladder inflammation and increased NF-kB/iNOS signaling. TRPA1 up-regulation and TRPV1/TRPV4 down-regulation may account for the MGO-induced bladder overactivity. Scavengers of MGO could be an option to ameliorate bladder dysfunction in diabetic conditions.

Long-term methylglyoxal intake aggravates murine Th2-mediated airway eosinophil infiltration.

Asthma outcomes is aggravated in obese patients. Excess of methylglyoxal (MGO) in obese/diabetic patients has been associated with diverse detrimental effects on cell function. This study aimed to evaluate the effects of long-term oral intake of MGO on ovalbumin-induced eosinophil inflammation. Male C57/Bl6 mice received 0.5% MGO in the drinking water for 12 weeks. Mice were sensitized and challenged with ovalbumin (OVA), and at 48 h thereafter, bronchoalveolar lavage (BAL) fluid and lungs were collected for cell counting, morphological analysis, and ELISA, mRNA expressions and DHE assays. In MGO-treated mice, OVA challenge significantly increased the peribronchiolar infiltrations of inflammatory cells and eosinophils compared with control group. Higher levels of IL-4, IL-5, and eotaxin in BAL fluid were also detected in MGO compared with control group. In addition, lung tissue of MGO-treated mice displayed significant increases in mRNA expressions of NF-κB and iNOS whereas COX-2 expression remained unchanged. The high TNF-α mRNA expression observed in lungs of OVA-challenged control mice was not further increased by MGO treatment. In MGO group, OVA-challenge increased significantly the NOX-2 and NOX-4 mRNA expressions, without affecting the NOX-1 expression. Levels of reactive-oxygen species (ROS) were significantly higher in lungs of MGO-treated mice, and no further increase by OVA-challenge was observed. In conclusion, 12-week intake of MGO exacerbates Th2-mediated airway eosinophil infiltration by activation of NF-kB/iNOS-dependent signaling pathway and positive regulation of NOX-2 and NOX-4 in the lung tissues. Scavengers of MGO could be an option to prevent obesity-related asthma.

Chronic ethanol consumption induces micturition dysfunction and alters the oxidative state of the urinary bladder.

Oxidative stress is pointed out as a major mechanism by which ethanol induces functional and structural changes in distinctive tissues. We evaluated whether ethanol consumption would increase oxidative stress and cause micturition dysfunction. Male C57BL/6J mice were treated with 20% ethanol (v/v) for 10 weeks. Our findings showed that chronic ethanol consumption reduced micturition spots and urinary volume in conscious mice, whereas in anaesthetized animals cystometric analysis revealed reduced basal pressure and increased capacity, threshold pressure, and maximum voiding. Treatment with ethanol reduced the contraction induced by carbachol in isolated bladders. Chronic ethanol consumption increased the levels of oxidant molecules and thiobarbituric acid reactive species in the mouse bladder. Upregulation of Nox2 was detected in the bladder of ethanol-treated mice. Increased activity of both superoxide dismutase and catalase were detected in the mouse bladder after treatment with ethanol. Conversely, decreased levels of reduced glutathione were detected in the bladder of ethanol-treated mice. The present study first demonstrated that chronic ethanol consumption induced micturition dysfunction and that this response was accompanied by increased levels of oxidant molecules in the mousebladder. These findings suggest that ethanol consumption is a risk factor for vesical dysfunction.

Menthol ameliorates voiding dysfunction in types I and II diabetic mouse model.

AIMS: Overactive bladder (OAB) is one of the most common complications of both type 1 (T1DM) and type 2 diabetes mellitus (T2DM). In healthy conditions, menthol infused intravesically reduces the threshold for initiating micturition reflex, but no study evaluated its effects in diabetic conditions. Therefore, we have used mouse models of T1DM and T2DM to evaluate the effects of menthol on cystometric alterations and increased bladder contractility in vitro. METHODS: For T1DM induction, male C57BL6 mice were injected with streptozotocin (STZ) and evaluated after 4 weeks. For T2DM induction, mice were fed with high-fat diet (HFD) for 12 weeks to induce obesity. Urodynamic profiles were assessed by filling cystometry through the infusion of menthol (100 µM for 30 min) or vehicle (DMSO 0.1%). Contractile responses to carbachol, potassium chloride (KCl), and electrical-field stimulation (EFS) were measured in isolated bladders after 20 min incubation with menthol (100 µM) or vehicle. RESULTS: Filling cystometry showed that STZ-injected mice exhibited higher bladder capacity, threshold pressure, and non-voiding contractions (NVCs), which were significantly reduced by menthol infusion. The increased voiding frequency in STZ group were unaffected by menthol. In HFD-fed obese mice menthol significantly attenuated the increased threshold pressure and NVC frequency, but unaffected the changes of voiding frequency. In both STZ-injected and HFD-fed mice, incubation of isolated bladders with menthol normalized the enhanced contractile responses to carbachol, KCl, and EFS stimulation. CONCLUSIONS: Menthol may be a potential pharmacological option for the treatment of OAB as a consequence of T1DM and T2DM.

Deletion or pharmacological blockade of TLR4 confers protection against cyclophosphamide-induced mouse cystitis.

Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a chronic inflammatory disease without consistently effective treatment. We investigate the role of toll-like receptor 4 (TLR4) on voiding dysfunction and inflammation in the cyclophosphamide (CYP)-induced mouse cystitis. Male C57BL/6 [wild-type, (WT)] and/or TLR4 knockout (TLR4-/-) mice were treated with an injection of CYP (300 mg/kg, 24 h) or saline (10 ml/kg). The pharmacological blockade of the TLR4 by resatorvid (10 mg/kg) was also performed 1 h prior CYP-injection in WT mice. Urodynamic profiles were assessed by voiding stain on filter paper and filling cystometry. Contractile responses to carbachol were measured in isolated bladders. In CYP-exposed WT mice, mRNA for TLR4, myeloid differentiation primary response 88, and TIR-domain-containing adapter-inducing interferon-ß increased by 45%, 72%, and 38%, respectively ( P < 0.05). In free-moving mice, CYP-exposed mice exhibited a higher number of urinary spots and smaller urinary volumes. Increases of micturition frequency and nonvoiding contractions, concomitant with decreases of intercontraction intervals and capacity, were observed in the filling cystometry of WT mice ( P < 0.05). Carbachol-induced bladder contractions were significantly reduced in the CYP group, which was paralleled by reduced mRNA for M2 and M3 muscarinic receptors. These functional and molecular alterations induced by CYP were prevented in TLR4-/- and resatorvid-treated mice. Additionally, the increased levels of inflammatory markers induced by CYP exposure, myeloperoxidase activity, interleukin-6, and tumor necrosis factor-alpha were significantly reduced by resatorvid treatment. Our findings reveal a central role for the TLR4 signaling pathway in initiating CYP-induced bladder dysfunction and inflammation and thus emphasize that TLR4 receptor blockade may have clinical value for IC/BPS treatment.

Phenotypic switching prevention and proliferation/migration inhibition of vascular smooth muscle cells by the ruthenium nitrosyl complex trans-[Ru(NO)Cl(cyclam](PF6 )2.

OBJECTIVES: Vascular smooth muscle cell (VSMC) migration and proliferation at sites of vascular injury are both critical steps in the development of intimal hyperplasia (IH). Local delivery of nitric oxide (NO) largely prevents these events. Among the NO donors, tetraazamacrocyclic nitrosyl complexes, such as trans-[Ru(NO)Cl(cyclam)](PF6 )2 (cyclamNO), gained attention for their features, which include the possibility of being embedded in solid matrices, and ability to participate in a nitrite/NO catalytic conversion cycle. METHODS: Methods used to evaluate cyclamNO activity: safety margin by NR and MTT; cell proliferation by 3H-thymidine incorporation and proliferating cell nuclear antigen (PCNA) expression; antimigratory properties by transwell and wound healing; prevention of cell phenotypic switching under platelet-derived growth factor type BB (PDGF-BB) stimuli by analysis of alpha smooth muscle actin (α-SMA) expression. KEY FINDINGS: Cell proliferation and migration induced by PDGF-BB were significantly inhibited by cyclamNO. The ~60% reduction on expression of contractile protein α-SMA induced by PDGF-BB revealed VSMC phenotypic switching which is significantly prevented by cyclamNO. Compared to the NO donor sodium nitroprusside, cyclamNO showed to be significantly less cytotoxic. CONCLUSIONS: With great potential to maintain VSMC functionality and prevent IH-associated events, cyclamNO might be a promissory drug for several applications in cardiovascular medicine, as in stents.

Micturition dysfunction in four-month old ovariectomized rats: Effects of testosterone replacement.

AIMS: Androgen deficiency has been implicated in urological complications of postmenopausal women. This study examined the effects of testosterone replacements on the lower urinary tract dysfunction in 4-month old ovariectomized (OVX) rats. MAIN METHODS: Sprague-Dawley female rats were OVX bilaterally. Three months later, rats received single intramuscular injections of testosterone undecanoate. Cystometric study, and bladder and urethra smooth muscle reactivities were evaluated. KEY FINDINGS: Ovariectomy reduced by 65% (p<0.05) the serum testosterone levels. Testosterone replacement at 5mg/kg restored serum hormone levels to baseline, whereas 10mg/kg produced 14-fold higher testosterone levels. OVX rats exhibited significant increases of body weight, perigonadal fat and blood pressure, and reduced uterus weight, but none of these parameters were changed by testosterone replacements. OVX rats exhibited micturition dysfunction characterized by increases of basal pressure, threshold pressure, voiding frequency and post-voiding pressure. In addition, the bladder contractions induced by electrical-field stimulation (EFS) and carbachol were significantly reduced, whereas angiotensin II-induced urethral contractions were significantly increased in OVX rats. Testosterone replacement at 10mg/kg (but not at 5mg/kg) dose fully normalized the in vivo micturition dysfunction, as well as the in vitro bladder and urethral alterations. Testosterone (10mg/kg) also significantly potentiated the bladder relaxations induced by the ß3-adrenoceptor agonist mirabegron. The protective effects of testosterone were not modified by concomitant treatment with the aromatase inhibitor letrozole (2.5mg/kg, 4weeks). SIGNIFICANCE: The improvement of micturition dysfunction by testosterone replacement suggests that androgen therapy might be of therapeutic benefit for urological complications associated with post-menopause.

Long-term treatment with the beta-3 adrenoceptor agonist, mirabegron ameliorates detrusor overactivity and restores cyclic adenosine monophosphate (cAMP) levels in obese mice.

AIMS: To evaluate the effects of the beta-3 adrenoceptor agonist, mirabegron in a mouse model of detrusor overactivity induced by obesity. METHODS: C57BL/6 male mice were fed with standard chow or high-fat diet for 12 weeks. Lean and obese mice were treated orally with mirabegron (10 mg/kg/day) from the last 2 weeks of diet. Cystometric evaluations, functional assays, protein expression for phosphodiesterase type 4 (PDE4), and cyclic adenosine monophosphate (cAMP) measurement were carried out. RESULTS: In obese mice the body weight, epididymal fat mass, fasting glucose, and low-density lipoprotein (LDL) levels were higher (P < 0.001) than in the lean mice. A reduction of 34% and 54% and an increase of 35% in the epididimal fat, LDL, and HDL levels (P < 0.05), respectively, were observed in the obese group treated with mirabegron, whereas no changes were seen in the lipid profile from lean mice. Obese group showed irregular micturition pattern, characterized by significant increases in frequency and non-void contractions. Carbachol, potassium chloride, and electrical-field stimulation induced detrusor smooth muscle (DSM) contractions, which were greater in bladders from obese mice than from lean mice. Two-week treatment with mirabegron restored all the contractile response alterations in the DSM. Basal intracellular levels of cAMP were reduced (68%), whereas PDE4 protein expression was increased (54%) in bladder from obese mice. Mirabegron restored the cAMP levels in obese bladder, without changing the PDE4 expression. CONCLUSION: Mirabegron was able to completely restore the urinary alterations seen in the bladder from obese mice.

New 2-Aminothiazoline derivatives lower blood pressure of spontaneously hypertensive rats (SHR) via I1-imidazoline and alpha-2 adrenergic receptors activation.

2-Aminothiazolines share an isosteric relationship with imidazolines and oxazolines with antihypertensive activity mainly mediated by the imidazoline I1-receptor. In the present work, we have prepared five aminothiazolines, following a previously described synthetic pathway. Aminothiazolines derived from dicyclopropylmethylamine (ATZ1) and cyclohexylamine (3) are unprecedented in the literature. Competitive radioligand assay was carried out with all synthetic compounds, and the I1 receptor affinity in comparison to rilmenidine in PC12 cells was determined. Surprisingly, the rilmenidine isoster (ATZ1) showed no I1-receptor interaction. Diethyl (ATZ4) and 2-ethyl-hexylamine (ATZ5) derivatives bind to the receptor with 11.98 and 10.94nmol/l, respectively. These compounds were selected for in vivo experiments. Both compounds reduced the blood pressure of spontaneously hypertensive rats (SHR). The hypotensive effect of these compounds was abrogated in the presence of α2 adrenergic (yohimbine) and I1 (efaroxan) receptor antagonists suggesting that both aminothiazolines bind to the adrenergic and imidazoline receptors. Lipinski's descriptors of the synthesized aminothiazolines were calculated and are similar to the known imidazoline I1 receptor ligands. 3D-Similarity between ATZ5 and agmatine, the natural imidazoline receptor ligand, was also observed.

Chronic treatment with resveratrol improves overactive bladder in obese mice via antioxidant activity.

The objective of the present work was to evaluate whether oral intake with resveratrol ameliorates overactive bladder in high-fat fed mice. Male C57BL6 mice fed with standard chow or high-fat diet to induce obesity received a two-week therapy with resveratrol (100mg/kg, given as a daily gavage). Weight and metabolic profile, together with cystometry and in vitro bladder contractions were evaluated. Measurements of gp91phox and SOD1 mRNA expressions and reactive-oxygen species (ROS) in bladder tissues, and serum TBARS were performed. Obese mice exhibited increases in body weight and epididymal fat mass, which were significantly reduced by oral treatment with resveratrol. Cystometric study in obese mice showed increases in non-voiding contractions, post-voiding pressure and voiding frequency that were reversed by resveratrol treatment. Likewise, the in vitro bladder overactivity in response to electrical-field stimulation (80V, 1-32Hz) or carbachol (1nM to 10mM) were normalized by resveratrol. The gp91phox and SOD1 mRNA expressions in bladder tissues were markedly higher in obese mice compared with lean group. In addition, ROS levels in bladder tissues and serum lipid peroxidation (TBARS assay) were markedly higher in obese compared with lean mice, all of which were reduced by resveratrol treatment. In lean group, resveratrol had no effect in any parameter evaluated. Our results show that two-week therapy of obese mice with resveratrol reduces the systemic and bladder oxidative stress, and greatly ameliorated the cystometry alterations and in vitro bladder overactivity. Resveratrol treatment could be an option to prevent obesity-associated overactive bladder.