Evidence that methylglyoxal and receptor for advanced glycation end products are implicated in bladder dysfunction of obese diabetic ob/ob mice.

Glycolytic overload in diabetes causes large accumulation of the highly reactive dicarbonyl compound methylglyoxal (MGO) and overproduction of advanced glycation end products (AGEs), which interact with their receptors (RAGE), leading to diabetes-associated macrovascular complications. The bladder is an organ that stays most in contact with dicarbonyl species, but little is known about the importance of the MGO-AGEs-RAGE pathway to diabetes-associated bladder dysfunction. Here, we aimed to investigate the role of the MGO-AGEs-RAGE pathway in bladder dysfunction of diabetic male and female ob/ob mice compared with wild-type (WT) lean mice. Diabetic ob/ob mice were treated with the AGE breaker alagebrium (ALT-711, 1 mg/kg) for 8 wk in drinking water. Compared with WT animals, male and female ob/ob mice showed marked hyperglycemia and insulin resistance, whereas fluid intake remained unaltered. Levels of total AGEs, MGO-derived hydroimidazolone 1, and RAGE in bladder tissues, as well as fluorescent AGEs in serum, were significantly elevated in ob/ob mice of either sex. Collagen content was also markedly elevated in the bladders of ob/ob mice. Void spot assays in filter paper in conscious mice revealed significant increases in total void volume and volume per void in ob/ob mice with no alterations of spot number. Treatment with ALT-711 significantly reduced the levels of MGO, AGEs, RAGE, and collagen content in ob/ob mice. In addition, ALT-711 treatment normalized the volume per void and increased the number of spots in ob/ob mice. Activation of AGEs-RAGE pathways by MGO in the bladder wall may contribute to the pathogenesis of diabetes-associated bladder dysfunction.NEW & NOTEWORTHY The involvement of methylglyoxal (MGO) and advanced glycation end products (AGEs) in bladder dysfunction of diabetic ob/ob mice treated with the AGE breaker ALT-711 was investigated here. Diabetic mice exhibited high levels of MGO, AGEs, receptor for AGEs (RAGE), and collagen in serum and/or bladder tissues along with increased volume per void, all of which were reduced by ALT-711. Activation of the MGO-AGEs-RAGE pathway in the bladder wall contributes to the pathogenesis of diabetes-associated bladder dysfunction.

The importance of the endothelial nitric oxide synthase on the release of 6-nitrodopamine from mouse isolated atria and ventricles and their role on chronotropism.

6-nitrodopamine (6-ND) is released from rat isolated atria, where it acts as a potent positive chronotropic agent. The release of 6-ND from rat isolated atria and ventricles is significantly reduced when pre-incubated with l-NAME, and the release was not affected by tetrodotoxin pre-treatment, indicating that in the heart, the origin of 6-ND is not neurogenic. Since l-NAME inhibits all three isoforms of NO synthase, it was investigated the basal release of 6-ND from isolated atria and ventricles from nNOS-/-, iNOS-/- and eNOS-/- mice of either sex. The release of 6-ND was measured by LC-MS/MS. There were no significant differences in the 6-ND basal release from isolated atria and ventricles from male control mice, as compared to female control mice. The 6-ND release from atria obtained from eNOS-/- mice was significantly reduced when compared to atria obtained from control mice. The 6-ND release in nNOS-/- mice was not significantly different compared to control animals whereas the 6-ND release from atria obtained from iNOS-/- mice was significantly higher when compared to control group. Incubation of the isolated atria with l-NAME caused a significant decrease in the basal atrial rate of control, nNOS-/-, and iNOS-/- mice, but not in eNOS-/- mice. The results clearly indicate that eNOS is the isoform responsible for the synthesis of 6-ND in the mice isolated atria and ventricles and supports the concept that 6-ND is the major mechanism by which endogenous NO modulates heart rate.

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.

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.

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.

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.

Activation of soluble guanylyl cyclase by BAY 58-2667 improves bladder function in cyclophosphamide-induced cystitis in mice.

Activators of soluble guanylyl cyclase (sGC) interact directly with its prosthetic heme group, enhancing the enzyme responsiveness in pathological conditions. This study aimed to evaluate the effects of the sGC activator BAY 58-2667 on voiding dysfunction, protein expressions of α1 and ß1 sGC subunits and cGMP levels in the bladder tissues after cyclophosphamide (CYP) exposure. Female C57BL/6 mice (20-25 g) were injected with CYP (300 mg/kg ip) to induce cystitis. Mice were pretreated or not with BAY 58-2667 (1 mg/kg, gavage), given 1 h before CYP injection. The micturition patterns and in vitro bladder contractions were evaluated at 24 h. In freely moving mice, the CYP injection produced reduced the micturition volume and increased the number of urine spots. Cystometric recordings in CYP-injected mice revealed significant increases in basal pressure, voiding frequency, and nonvoiding contractions (NVCs), along with decreases in bladder capacity, intercontraction interval, and compliance. BAY 58-2667 significantly prevented the micturition alterations observed in both freely moving mice and cystometry and normalized the reduced in vitro carbachol-induced contractions in the CYP group. Reduced protein expressions of α1 and ß1 sGC subunits and of cGMP levels were observed in the CYP group, all of which were prevented by BAY 58-2667. CYP exposure significantly increased reactive-oxygen species (ROS) generation in both detrusor and urothelium, and this was normalized by BAY 58-2667. The increased myeloperoxidase and cyclooxygenase-2 activities in the bladders of the CYP group remained unchanged by BAY 58-2667. Activators of sGC may constitute a novel and promising therapeutic approach for management of interstitial cystitis.

The beta-3 adrenoceptor agonist, mirabegron relaxes isolated prostate from human and rabbit: new therapeutic indication?

BACKGROUND: Alpha1 (α1)-blockers, 5-alpha reductase and phosphodiesterase type-5 inhibitors are pharmacological classes currently available for benign prostatic hyperplasia (BPH) treatment. Mirabegron, a beta-3 adrenoceptor (ß3-AR) agonist has been approved for the therapy of overactive bladder and may constitute a new therapeutic option for BPH treatment. This study is aimed to evaluate the in vitro effects of mirabegron in human and rabbit prostatic smooth muscle. METHODS: In rabbit prostate, electrical field stimulation (EFS)-induced contraction and concentration-response curve (CRC) to mirabegron in phenylephrine pre-contracted tissues were carried out. The potency (pEC50 ) and maximal response (Emax ) values were determined. In human prostate, CRC to phenylephrine was carried out in the absence and presence of mirabegron. Immunohistochemistry analysis for ß3-AR was also carried out. RESULTS: In human prostate, immunohistochemistry analysis revealed the presence of ß3-AR on the transition zone and mirabegron reduced by 42% the phenylephrine-induced contractions. In rabbit prostate, mirabegron produced concentration-dependent relaxations (pEC50 : 6.01 ± 0.12; Emax : 106 ± 3%), which were fully resistant to the blockade of ß1-AR and ß2-AR. The ß3-AR blocker L748,337 caused a six-fold rightward shift in mirabegron-induced relaxations. Mirabegron (10 µM) reduced by 63% the EFS-induced contractions. Inhibitors of nitric oxide (L-NAME) and of soluble guanylate cyclase (ODQ) along with a cocktail of K+ channel blockers (apamin, charybdotoxin, glibenclamide, tetraethylammonium) all failed to significantly affect the mirabegron-induced rabbit relaxations. CONCLUSION: Mirabegron relaxes prostatic smooth muscle, providing an experimental support for the clinical investigation of its combination with an α1-blockers or PDE5 inhibitors in the treatment of BPH. Prostate 75:440-447, 2015. © 2014 Wiley Periodicals, Inc.

Pharmacological characterisation of the relaxation induced by the soluble guanylate cyclase activator, BAY 60-2770 in rabbit corpus cavernosum.

OBJECTIVE: To characterise the relaxation induced by the soluble guanylate cyclase (sGC) activator, BAY 60-2770 (4-({(4-carboxybutyl) [2- (5-fluoro-2-{[4'-(trifluoromethyl) biphenyl-4-yl]methoxy}phenyl)ethyl] amino}methyl)benzoic acid) in rabbit corpus cavernosum (CC). MATERIAL AND METHODS: The penis from male New Zealand rabbits was removed and fours strips of CC were obtained. Concentration-response curves to BAY 60-2770 were constructed in the absence and presence of inhibitors of nitric oxide synthase, N (G)-nitro-L- arginine methyl ester (L-NAME, 100 µm), sGC, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 µm) and phosphodiesterase type 5 (PDE-5), tadalafil (0.1 µm). The potency (pEC50 ) and maximal response (Emax ) values were determined. Then, electrical-field stimulation (EFS)-induced contraction or relaxation was tested in the absence and presence of BAY 60-2770 (0.1 or 1 µm) alone or combined with ODQ (10 µm). For EFS-induced relaxation two protocols were used: (i) ODQ (10 µm) was first incubated for 20 min and then BAY 60-2770 (1 µm) was added for another 20 min (ODQ + BAY 60-2770); (ii) in different CC strips, BAY 60-2770 was incubated for 20 min followed by another 20 min with ODQ (BAY 60-2770 + ODQ). The intracellular levels of cyclic guanosine monophosphate (cGMP) were also determined. RESULTS: BAY 60-2770 potently relaxed rabbit CC with mean (sem) pEC50 and Emax values of 7.58 (0.19) and 81 (4)%, respectively. The inhibitors ODQ (n = 7) or tadalafil (n = 7) produced 4.2- and 6.3-leftward shifts, respectively in BAY 60-2770-induced relaxation without interfering with the Emax values. The intracellular levels of cGMP were augmented after stimulation with BAY 60-2770 (1 µm) alone, whereas its co-incubation with ODQ produced even higher levels of cGMP. The EFS-induced contraction was reduced in the presence of BAY 60-2770 (1 µm) and this inhibition was even greater when BAY 60-2770 was co-incubated with ODQ. The nitrergic stimulation induced CC relaxation, which was abolished in the presence of ODQ. BAY 60-2770 alone increased the amplitude of relaxation. Co-incubation of ODQ and BAY 60-2770 did not alter the relaxation in comparison with ODQ alone. Interestingly, when BAY 60-2770 was incubated before ODQ, EFS-induced relaxation was partly restored in comparison with ODQ alone or ODQ + BAY 60-2770. CONCLUSIONS: The relaxation induced by the sGC activator, BAY 60-2770 was increased after sGC oxidation and unaltered in the absence of nitric oxide. Thus, this class of substances may have advantages over sGC stimulators or PDE-5 inhibitors for treating patients with erectile dysfunction and extensive endothelial damage.

Soluble guanylyl cyclase (sGC) degradation and impairment of nitric oxide-mediated responses in urethra from obese mice: reversal by the sGC activator BAY 60-2770.

Obesity has emerged as a major contributing risk factor for overactive bladder (OAB), but no study examined urethral smooth muscle (USM) dysfunction as a predisposing factor to obesity-induced OAB. This study investigated the USM relaxant machinery in obese mice and whether soluble guanylyl cyclase (sGC) activation with BAY 60-2770 [acid 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4-(trifluoromethyl) biphenyl-4-yl] methoxy} phenyl) ethyl] amino} methyl) benzoic] rescues the urethral reactivity through improvement of sGC-cGMP (cyclic guanosine monophosphate) signaling. Male C57BL/6 mice were fed for 12 weeks with a high-fat diet to induce obesity. Separate groups of animals were treated with BAY 60-2770 (1 mg/kg per day for 2 weeks). Functional assays and measurements of cGMP, reactive-oxygen species (ROS), and sGC protein expression in USM were determined. USM relaxations induced by NO (acidified sodium nitrite), NO donors (S-nitrosoglutathione and glyceryl trinitrate), and BAY 41-2272 [5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine] (sGC stimulator) were markedly reduced in obese compared with lean mice. In contrast, USM relaxations induced by BAY 60-2770 (sGC activator) were 43% greater in obese mice (P < 0.05), which was accompanied by increases in cGMP levels. Oxidation of sGC with ODQ [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one] (10 µM) potentiated BAY 60-2770-induced USM responses in the lean group. Long-term oral BAY 60-2770 administration fully prevented the impairment of USM relaxations in obese mice. Reactive-oxygen species (ROS) production was enhanced, but protein expression of ß1 second guanylate cyclase subunit was reduced in USM from obese mice, both of which were restored by BAY 60-2770 treatment. In conclusion, impaired USM relaxation in obese mice is associated with ROS generation and down-regulation of sGC-cGMP signaling. Prevention of sGC degradation by BAY 60-2770 ameliorates the impairment of urethral relaxations in obese mice.

The soluble guanylyl cyclase activator BAY 60-2770 ameliorates overactive bladder in obese mice.

PURPOSE: Activators of soluble guanylyl cyclase are of potential interest as treatment for cardiovascular diseases but to our knowledge they have never been proposed to treat overactive bladder. We evaluated the effects of the soluble guanylyl cyclase activator BAY 60-2270 on voiding dysfunction and detrusor overactivity in a mouse model of obesity associated overactive bladder. MATERIALS AND METHODS: C57BL/6 male mice fed for 10 weeks with standard chow or a high fat diet were treated with 1 mg/kg BAY 60-2770 per day for 2 weeks via gavage. Cystometric evaluations were done and responses to contractile agents in isolated bladders were determined. RESULTS: Obese mice showed an irregular micturition pattern characterized by significant increases in voiding and nonvoiding contractions, which were normalized by BAY 60-2770. Carbachol, KCl and CaCl2 produced concentration dependent contractions in isolated bladder strips, which were markedly greater in obese than in lean mice. BAY 60-2770 normalized bladder contractions in the obese group. A 78% increase in reactive oxygen species generation in the bladder tissue of obese mice was observed, which was unaffected by BAY 60-2770. Treatment with BAY 60-2770 generated a tenfold increase in cyclic guanosine monophosphate in the bladders of obese mice without affecting the nucleotide level in the lean group. Protein expression of the soluble guanylyl cyclase α1 and ß1 subunits was decreased 40% in the bladder tissue of obese mice but restored by BAY 60-2770. CONCLUSIONS: Two-week BAY 60-2770 therapy increased cyclic guanosine monophosphate and rescued expression of the soluble guanylyl cyclase α1 and ß1 subunits in bladder tissue, resulting in great amelioration of bladder dysfunction.

Prolonged therapy with the soluble guanylyl cyclase activator BAY 60-2770 restores the erectile function in obese mice.

INTRODUCTION: Cardiovascular and endocrine-metabolic diseases associated with increased oxidative stress such as obesity lead to erectile dysfunction (ED). Activators of soluble guanylyl cyclase (sGC) such as BAY 60-2770 reactivate the heme-oxidized sGC in vascular diseases. AIM: This study aimed to evaluate the effects of 2-week oral intake with BAY 60-2270 on a murine model of obesity-associated ED. METHODS: C57BL/6 male mice were fed for 12 weeks with standard chow or high-fat diet. Lean and obese mice were treated with BAY 60-2770 (1 mg/kg/day, 2 weeks). MAIN OUTCOME MEASURES: Measurements of intracavernosal pressure (ICP), along with acetylcholine (10(-9) to 10(-5) M) and electrical field stimulation (EFS; 4-10 Hz)-induced corpus cavernosum relaxations in vitro, were obtained. Levels of cyclic guanosine monophosphate (cGMP), reactive oxygen species (ROS), and sGC protein expressions in cavernosal tissues were measured. RESULTS: Cavernous nerve stimulation caused frequency-dependent ICP increases, which were significantly lower in obese compared with lean mice (P < 0.05). Two-week therapy with BAY 60-2770 fully reversed the decreased ICP in obese group. Acetylcholine-induced cavernosal relaxations were 45% lower (P < 0.001) in obese mice, which were fully restored by BAY 60-2770 treatment. Likewise, the EFS-induced relaxations in obese mice were restored by BAY 60-2770. Basal cGMP content in erectile tissue was 68% lower (P < 0.05) in obese mice, an effect normalized by BAY 60-2770. Levels of ROS were 52% higher (P < 0.05) whereas protein expression of α1 sGC subunit was reduced in cavernosal tissue of obese mice, both of which were normalized by BAY 60-2770. In lean group, BAY 60-2770 did not significantly affect any functional, biochemical, or molecular parameter analyzed. CONCLUSIONS: Two-week therapy with BAY 60-2770 restores the erectile function in obese mice that is associated with reduced ROS levels, up-regulation of α1 sGC subunit, and increased cGMP levels in the erectile tissue.

6-Nitrodopamine is an endogenous mediator of the rabbit corpus cavernosum relaxation.

BACKGROUND: 6-Nitrodopamine (6-ND) is a novel endogenous catecholamine that has a potent relaxant action on vascular smooth muscle in vitro. OBJECTIVES: To evaluate the basal release of 6-ND and noradrenaline from rabbit-isolated corpus cavernosum (RbCC) and its relaxing action on this tissue. METHODS: Rabbit corpus cavernosa were dissected and suspended in a 5-mL organ bath containing oxygenated Krebs-Henseleit's solution. 6-ND and noradrenaline release was quantified by liquid chromatography coupled to tandem mass spectrometry. The relaxant activity of 6-ND was assessed in RbCC strips pre-contracted with endothelin-1 (10 nM). RESULTS: Rabbit corpus cavernosum presented basal release of both 6-ND (2.9 ± 0.8 ng/mL, n = 12) and noradrenaline (1.7 ± 1.3 ng/mL, n = 12). The 6-ND release was reduced by pre-treatment with Nω-nitro-l-arginine methyl ester (l-NAME) (100 µM), whereas that of noradrenaline was unaffected. Tetrodotoxin (TTX, 1 µM) abolished the noradrenaline release but had no effect on 6-ND release, indicating a non-neurogenic origin for 6-ND. 6-ND and the selective dopamine D2-agonist L-741,626 caused concentration-dependent RbCC relaxations (pEC50 of 11 ± 0.15 and 11.15 ± 0.28, respectively). Pre-treatment with either l-NAME or the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-on (ODQ) (100 µM) caused a rightward shift of the concentration-response curve to 6-ND, without affecting the L-741,626 responses. In TTX (100 nM)-pre-treated preparations, neither l-NAME nor ODQ shifted the 6-ND concentration-response curve. Dopamine, noradrenaline, and adrenaline caused concentration-dependent RbCC contractions. Pre-incubation with 6-ND concentration-dependently inhibited the dopamine-induced contractions, without affecting those induced by either noradrenaline or adrenaline. DISCUSSION AND CONCLUSION: 6-Nitrodopamine is the most potent endogenous relaxant agent in RbCC ever described and represents a novel mechanism by which NO causes corpus cavernosum smooth muscle relaxation. The finding that 6-ND acts as a truly selective dopamine D2-receptor antagonist indicates that the balance of dopamine and 6-ND release/synthesis may be the main mechanism that modulates corpus cavernosum smooth muscle tonus in vivo.

Basal release and relaxation responses to 6-nitrodopamine in swine carotid, coronary, femoral, and renal arteries.

Mammalian and reptilian vascular tissues present basal release of 6-nitrodopamine, which is reduced when the tissues are pre-incubated with the NO synthase inhibitor L-NG-Nitro arginine methyl ester (L-NAME), or when the endothelium is mechanically removed. 6-Nitrodopamine induces vasorelaxation in pre-contracted vascular rings by antagonizing the dopaminergic D2-like receptor. Here it was investigated whether male swine vessels (including carotid, left descendent coronary, renal, and femoral arteries) release 6-nitrodopamine, dopamine, noradrenaline, and adrenaline, as measured by liquid chromatography coupled to tandem mass spectrometry. The in vitro vasorelaxant action of 6-nitrodopamine was evaluated in carotid, coronary, renal, and femoral arteries precontracted by U-46619 (3 nM), and compared to that induced by the dopamine D2-receptor antagonist L-741,626. Expression of tyrosine hydroxylase and the neuromaker calretinin was investigated by immunohistochemistry. All vascular tissues presented basal release of endothelium-derived catecholamines. The relaxation induced by 6-nitrodopamine was not affected by preincubation of the tissues with either L-NAME (100 µM, 30-min preincubation) or the heme-site inhibitor of soluble guanylyl cyclase ODQ (100 µM, 30-min preincubation). Electrical field stimulation (EFS)-induced contractions were significantly potentiated by previous incubation with L-NAME, but unaffected by ODQ preincubation. The contractions induced by EFS were reduced by preincubation with either 6-nitrodopamine or L-741,626. Immunohistochemistry in all arteries revealed the presence of tyrosine hydroxylase in the endothelium, whereas immunoreactivity for calretinin was negative. Swine vessels present basal release of endothelium-derived catecholamines and expression of tyrosine hydroxylase in the endothelium. The vasodilation induced by 6-nitrodopamine is due to blockade of dopaminergic D2-like receptors.

Insulin relaxes bladder via PI3K/AKT/eNOS pathway activation in mucosa: unfolded protein response-dependent insulin resistance as a cause of obesity-associated overactive bladder.

We aimed to investigate the role of insulin in the bladder and its relevance for the development of overactive bladder (OAB) in insulin-resistant obese mice. Bladders from male individuals who were involved in multiple organ donations were used. C57BL6/J mice were fed with a high-fat diet for 10 weeks to induce insulin-resistant obesity. Concentration-response curves to insulin were performed in human and mouse isolated mucosa-intact and mucosa-denuded bladders. Cystometric study was performed in terminally anaesthetized mice. Western blot was performed in bladders to detect phosphorylated endothelial NO synthase (eNOS) (Ser1177) and the phosphorylated protein kinase AKT (Ser473), as well as the unfolded protein response (UPR) markers TRIB3, CHOP and ATF4. Insulin (1-100 nm) produced concentration-dependent mouse and human bladder relaxations that were markedly reduced by mucosal removal or inhibition of the PI3K/AKT/eNOS pathway. In mouse bladders, insulin produced a 3.0-fold increase in cGMP levels (P < 0.05) that was prevented by PI3K/AKT/eNOS pathway inhibition. Phosphoinositide 3-kinase (PI3K) inhibition abolished insulin-induced phosphorylation of AKT and eNOS in bladder mucosa. Obese mice showed greater voiding frequency and non-voiding contractions, indicating overactive detrusor smooth muscle. Insulin failed to relax the bladder or to increase cGMP in the obese group. Insulin-stimulated AKT and eNOS phosphorylation in mucosa was also impaired in obese mice. The UPR markers TRIB3, CHOP and ATF4 were increased in the mucosa of obese mice. The UPR inhibitor 4-phenyl butyric acid normalized all the functional and molecular parameters in obese mice. Our data show that insulin relaxes human and mouse bladder via activation of the PI3K/AKT/eNOS pathway in the bladder mucosa. Endoplasmic reticulum stress-dependent insulin resistance in bladder contributes to OAB in obese mice.

Superoxide anion production by NADPH oxidase plays a major role in erectile dysfunction in middle-aged rats: prevention by antioxidant therapy.

INTRODUCTION.: Prevalence of erectile dysfunction (ED) increases progressively with aging, but the ED pathophysiology at its early stages is still poorly investigated. AIM.: This study aimed to evaluate the functional and molecular alterations of erectile function at middle age, focusing on the contribution of oxidative stress in erectile tissue for the ED. METHODS.: Young (3.5-month) and middle-aged (10-month) male Wistar rats were used. Rat corpus cavernosum (RCC) was dissected free and mounted in 10-mL organ baths containing Krebs solution. Intracavernosal pressure (ICP) in anesthetized rats was evaluated. MAIN OUTCOME MEASURES.: Concentration-response curves to endothelium-dependent and endothelium-independent agents, as well as to electrical field stimulation (EFS), were obtained in RCC strips. Measurement of cyclic guanosine monophosphate (cGMP) and expressions of neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS), gp91(phox) and superoxide dismutase-1 (SOD-1) expressions in RCC were evaluated. RESULTS.: ICP was significantly reduced in middle-aged compared with young rats. RCC relaxations to acetylcholine (10(-8) to 10(-2) M), sodium nitroprusside (10(-8) to 10(-2) M), sildenafil (10(-9) to 10(-5) M), BAY 41-2272 (10(-9) to 10(-5) M), and EFS (4-32 Hz) were decreased in middle-aged group, which were nearly normalized by apocynin (NADPH oxidase inhibitor; 10(-4) M) or SOD (75 U/mL). Prolonged treatment with apocynin (85 mg/rat/day, 4 weeks) also restored the impaired relaxations in middle-aged rats. Relaxations to 8-bromoguanosine 3',5'-cyclic monophosphate sodium salt (8-Br-cGMP; 10(-8) to 3 × 10(-4) M) remained unchanged between groups. Basal and stimulated cGMP production were lower in middle-aged group, an effect fully restored by apocynin and SOD. Protein expression of nNOS and phosphorylated eNOS (p-eNOS) (Ser-1177) reduced, whereas gp(91phox) mRNA expression increased in RCC from middle-aged rats. CONCLUSIONS.: ED in middle-aged rats is associated with decreased NO bioavailability in erectile tissue due to upregulation of NADPH oxidase subunit gp91(phox) and downregulation of nNOS/p-eNOS. Antioxidant therapies may be a good pharmacological approach to prevent ED at its early stages.

Investigation on the positive chronotropic action of 6-nitrodopamine in the rat isolated atria.

6-Nitrodopamine (6-ND) is released from rat isolated atria being 100 times more potent than noradrenaline and adrenaline, and 10,000 times more potent than dopamine as a positive chronotropic agent. The present study aimed to investigate the interactions of 6-ND with the classical catecholamines, phosphodiesterase (PDE)-3 and PDE4, and the protein kinase A in rat isolated atria. Atrial incubation with 1 pM of dopamine, noradrenaline, or adrenaline had no effect on atrial frequency. Similar results were observed when the atria were incubated with 0.01 pM of 6-ND. However, co-incubation of 6-ND (0.01 pM) with dopamine, noradrenaline, or adrenaline (1 pM each) resulted in significant increases in atrial rate, which persisted over 30 min after washout of the agonists. The increased atrial frequency induced by co-incubation of 6-ND with the catecholamines was significantly reduced by the voltage-gated sodium channel blocker tetrodotoxin (1 µM, 30 min), indicating that the positive chronotropic effect of 6-ND is due in part to activation of nerve terminals. Pre-treatment of the animals with reserpine had no effect on the positive chronotropic effect induced by dopamine, noradrenaline, or adrenaline; however, reserpine markedly reduced the 6-ND (1 pM)-induced positive chronotropic effect. Incubation of the rat isolated atria with the protein kinase A inhibitor H-89 (1 µM, 30 min) abolished the increased atrial frequency induced by dopamine, noradrenaline, and adrenaline, but only attenuated the increases induced by 6-ND. 6-ND induces catecholamine release from adrenergic terminals and increases atrial frequency independently of PKA activation.

TRPA1 channel mediates methylglyoxal-induced mouse bladder dysfunction.

Introduction: The transient receptor potential ankyrin 1 channel (TRPA1) is expressed in urothelial cells and bladder nerve endings. Hyperglycemia in diabetic individuals induces accumulation of the highly reactive dicarbonyl compound methylglyoxal (MGO), which modulates TRPA1 activity. Long-term oral intake of MGO causes mouse bladder dysfunction. We hypothesized that TRPA1 takes part in the machinery that leads to MGO-induced bladder dysfunction. Therefore, we evaluated TRPA1 expression in the bladder and the effects of 1 h-intravesical infusion of the selective TRPA1 blocker HC-030031 (1 nmol/min) on MGO-induced cystometric alterations. Methods: Five-week-old female C57BL/6 mice received 0.5% MGO in their drinking water for 12 weeks, whereas control mice received tap water alone. Results: Compared to the control group, the protein levels and immunostaining for the MGO-derived hydroimidazolone isomer MG-H1 was increased in bladders of the MGO group, as observed in urothelium and detrusor smooth muscle. TRPA1 protein expression was significantly higher in bladder tissues of MGO compared to control group with TRPA1 immunostaining both lamina propria and urothelium, but not the detrusor smooth muscle. Void spot assays in conscious mice revealed an overactive bladder phenotype in MGO-treated mice characterized by increased number of voids and reduced volume per void. Filling cystometry in anaesthetized animals revealed an increased voiding frequency, reduced bladder capacity, and reduced voided volume in MGO compared to vehicle group, which were all reversed by HC-030031 infusion. Conclusion: TRPA1 activation is implicated in MGO-induced mouse overactive bladder. TRPA1 blockers may be useful to treat diabetic bladder dysfunction in individuals with high MGO levels.

Alpha1-adrenergic antagonists block 6-nitrodopamine contractions on the rat isolated epididymal vas deferens.

6-nitrodopamine (6-ND) is released from rat isolated vas deferens and modulates electrical-field stimulation (EFS) contractions of the rat isolated epididymal vas deferens (RIEVD) via a specific receptor which is blocked by tricyclic antidepressants. Here, the effects of selective α1-adrenergic receptor antagonists on RIEVD contractions induced by 6-ND, dopamine, noradrenaline, adrenaline and EFS were investigated. Doxazosin and tamsulosin (3-10 nM) caused significant rightward shifts of the concentration-response curve to 6-ND, but had no effect on dopamine-, noradrenaline- and adrenaline-induced contractions. Alfuzosin (10 nM) produced rightward shifts on concentration-response curves to all catecholamines. Silodosin (10 nM) and terazosin (100 nM) displaced to the right the noradrenaline, dopamine and adrenaline curves, but higher concentrations of both antagonists (100 and 300 nM, respectively) were required to displace the 6-ND curves. The EFS-induced contractions were significantly inhibited only at the concentrations that the α1-adrenergic receptor antagonists caused rightward shifts on the 6-ND concentration-response curves. The inhibition of EFS-induced contractions by doxazosin (10 nM), tamsulosin (10 nM), alfuzosin (10 nM), silodosin (100 nM) and terazosin (300 nM), were not observed in RIEVD obtained from animals chronically treated with L-NAME. This work demonstrates that α1-adrenoceptor antagonists act as 6-ND receptor antagonists in RIEVD, opening the possibility that many actions previously attributed to noradrenaline could be due to 6-ND antagonism. In addition, blockade of the 6-ND receptors by both tricyclic antidepressants and α1-adrenergic receptor antagonists may represent the common mechanism of action responsible for their therapeutic use in the treatment of premature ejaculation.

ß1- and ß1/ß2-adrenergic receptor antagonists block 6-nitrodopamine-induced contractions of the rat isolated epididymal vas deferens.

6-Nitrodopamine (6-ND) is an endogenous modulator of the contractility in the rat isolated epididymal vas deferens (RIEVD) and considered to be the main peripheral mediator of the emission process. Use of selective and unselective ß-adrenergic receptor antagonists has been associated with ejaculatory failure. Here, the effects of selective ß1- and ß1/ß2-adrenergic receptor antagonists on RIEVD contractions induced by 6-ND, dopamine, noradrenaline, adrenaline, and electric-field stimulation (EFS) were investigated. The selective ß1-adrenergic receptor antagonists atenolol (0.1 and 1 µï»¿M), betaxolol (1 µï»¿M), and metoprolol (1 µï»¿M) and the unselective ß1/ß2-adrenergic receptor antagonists propranolol (1 and 10 µï»¿M) and pindolol (10 µï»¿M) caused significant rightward shifts of the concentration-response curve to 6-ND (pA2 6.41, 6.91, 6.75, 6.47, and 5.74; for atenolol, betaxolol, metoprolol, propranolol, and pindolol), but had no effect on dopamine-, noradrenaline-, and adrenaline-induced contractions. The effects of selective ß1- and ß1/ß2-adrenergic receptor antagonists at a higher concentration (atenolol 1 µï»¿M, betaxolol 1 µï»¿M, metoprolol 1 µï»¿M, propranolol 10 µï»¿M, and pindolol 10 µï»¿M) also reduced the EFS-induced RIEVD contractions in control, but not in RIEVD obtained from L-NAME-treated animals. The selective ß1-adrenoceptor agonist RO-363, the selective ß2-adrenoceptor agonist salbutamol, and the selective ß3-adrenoceptor agonist mirabegron, up to 300 µï»¿M, had no effect on the RIEVD tone. The results demonstrate that ß1- and ß1-/ß2-adrenoceptor receptor antagonists act as 6-ND receptor antagonists in RIEVD, further confirming the main role of 6-ND in the RIEVD contractility.