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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 α 1 -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 α 1 -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 α 1 -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 α 1 -adrenergic receptor antagonists may represent the common mechanism of action responsible for their therapeutic use in the treatment of premature ejaculation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

6-nitrodopamine (6-ND) is released from human umbilical cord vessels and modulates vascular reactivity by acting as a dopamine antagonist. Here we investigate whether 6-ND is released by the rat isolated vas deferens and its effect on this tissue. Dopamine, noradrenaline, adrenaline and 6-ND levels were quantified in rat isolated vas deferens by LC-MS-MS. Electric-field stimulation (EFS) and concentration-response curves to 6-ND, noradrenaline, dopamine and adrenaline were performed in the absence and in the presence (30 min) of L-NAME, SCH-23390, haloperidol, PG-01037, sonepiprazole, desipramine, clomipramine, amitriptyline, cyclobenzaprine, carbamazepine, maprotiline, paroxetine, oxcarbazepine and ketanserin in the rat isolated epididymal vas deferens (RIEVD). Basal releases of 6-ND and noradrenaline were detected from the rat isolated vas deferens. 6-ND release was reduced by tissue incubation with L-NAME and from the vas deferens obtained from L-NAME-treated rats. SCH-23390 caused leftward shifts on concentration-response curves to 6-ND without affecting dopamine- or EFS-induced RIEVD contractions. Haloperidol, PG-01037 and sonepiprazole caused significant rightward shifts on concentration-response curves to dopamine but had no effect on either the 6-ND or EFS-induced RIEVD contractions. The tricyclic compounds desipramine, clomipramine, amitriptyline, cyclobenzaprine and carbamazepine induced rightward shifts on 6-ND concentration-response curve but did not reduce the noradrenaline, dopamine and adrenaline contractile responses. They also reduced the EFS-induced RIEVD contractions in control but not in tissues obtained from L-NAME-treated animals. Maprotiline, oxcarbazepine, paroxetine and ketanserin had no effect in either 6-ND or EFS-induced RIEVD contractions. Thus, 6-ND modulates RIEVD contractility, and desipramine, clomipramine, amitriptyline, cyclobenzaprine and carbamazepine act as selective 6-ND receptor antagonists., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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,β-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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mirabegron is the first β3-adrenoceptor agonist approved on the market and may offer beneficial pharmacological action in patients with overactive bladder and erectile dysfunction. Here, we further investigate the mechanisms by which mirabegron induces rat corpus cavernosum (CC) relaxation. Adult male Wistar rats were used. The CC were isolated for in vitro functional assays and β-adrenoceptors subtypes mRNA expression evaluation. Animals were treated orally with mirabegron (30 mg/kg, 3 h), tadalafil (10 mg/kg, 3 h) or both for intracavernous pressure (ICP). Intracellular levels of cAMP and cGMP were also determined. The β 1 -, β 2 - and β 3 -adrenoceptors subtypes were expressed in rat CC. Mirabegron produced concentration-dependent CC relaxations that were unaffected by the β 1 -, β 2 - or β 3 -adrenoceptor antagonists atenolol (1 μM), ICI-118,551 (1 μM) and L748,337 (10 μM), respectively. Mirabegron-induced relaxations were not affected by the phosphodiesterase type 4 inhibitor, rolipram, or the adenylyl cyclase selective inhibitor, SQ 22,536. Potassium channel- or calcium influx-blockade are not involved in mirabegron-induced relaxations. In contrast, mirabegron produced rightward shifts in the contractile response induced by the α1-adrenoceptor agonist, phenylephrine. Finally, cavernous nerve stimulation caused frequency-dependent ICP increases, which were significantly increased in rats treated with mirabegron in a similar degree of tadalafil-treated rat, without promoting a significant cAMP or cGMP accumulation. Together, our results demonstrate that mirabegron induced CC relaxation through α1-adrenoceptor blockade. Care should be taken to translate the effect of mirabegron into the clinic, especially when using rat as an animal model of erectile dysfunction., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Obese mice display overactive bladder (OAB) associated with impaired urethra smooth muscle (USM) function. In this study, we evaluated the role of the adipose tissue surrounding the urethra and prostate in obese mice (here referred as periprostatic adipose tissue; PPAT) to the USM dysfunction. Male C57BL6/JUnib mice fed with either a standard-chow or high-fat diet to induce obesity were used. In PPAT, histological analysis, and qPCR analysis for gp91phox, tumor necrosis factor-α (TNF-α) and superoxide dismutase (SOD) were conducted. In USM, concentration-response curves to contractile and relaxing agents, as well as measurements of reactive-oxygen species and nitric oxide (NO) levels were performed. The higher PPAT area in obese mice was accompanied by augmented gp91phox (NOX2) and TNF-α expressions, together with decreased SOD1 expression. In USM of obese group, the contractile responses to phenylephrine and vasopressin were increased, whereas the relaxations induced with glyceryl trinitrate were reduced. The reactive-oxygen species and NO levels in USM of obese mice were increased and decreased, respectively. A higher SOD expression was also detected in obese group whilst no changes in the gp91phox levels were observed. We next evaluated the effects of the antioxidant resveratrol (100 mg/kg/day, two-weeks, PO) in the functional alterations and NO levels of obese mice. Resveratrol treatment in obese mice reversed both the functional USM dysfunction and the reduced NO production. Our data show that PPAT exerts a local inflammatory response and increases oxidative stress that lead to urethral dysfunction. Resveratrol could be an auxiliary option to prevent obesity-associated urethral dysfunction., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mirabegron is a β 3 -adrenoceptor agonist and released on the marked for the treatment of overactive bladder. Because mirabegron is the only β 3 -adrenoceptor agonist available and substances that increase the levels of cyclic adenosine monophosphate (cAMP) inhibit platelet activity, we tested the hypothesis that mirabegron could have antiplatelet activity. Collagen- and thrombin induced platelet aggregation, thromboxane B2 (TXB 2 ) and cyclic nucleotides quantification and calcium (Ca 2+ ) mobilization were determined in the absence and presence of mirabegron in human washed platelets. Our results revealed that mirabegron (10-300 µM) produced significant inhibitions on platelet aggregation induced by collagen- or thrombin, accompanied by greater intracellular levels of cAMP. The β 3 -adrenoceptor antagonist L 748,337 (1 µM) and the adenylate cyclase inhibitor, SQ 22,536 (100 µM) reversed the inhibition induced by mirabegron in thrombin-stimulated platelets. The selective antagonists for β 1 -and β 2 -adrenoceptors, atenolol and ICI 117,551 (3 µM), respectively did not interfere on the inhibition induced by mirabegron. In Fluo-4 loaded platelets, mirabegron reduced the total and intracellular Ca 2+ levels. Pre-incubation with mirabegron almost abolished the levels of TXB2. Mirabegron did not augment the intracellular levels of cyclic guanosine monophosphate. In conclusion, mirabegron inhibited human platelet aggregation through cAMP accumulation, thus suggesting that substances that activate β 3 -adrenoceptor could be beneficial as adjuvant antiplatelet therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Impairment of nitric oxide (NO) - cyclic GMP signaling pathway is likely to contribute to human begnin prostate hyperplasia (BPH). In the present study we have used a model of chronic NO synthesis inhibition to evaluate the functional alterations of prostate smooth muscle (PSM) machinery, and involvement of Rho-kinase pathway. Wistar rats were treated with the NO inhibitor N(ω)-nitro-l-arginine methyl ester (L-NAME, 20mg/kg/day; 4 weeks), after which contractile responses to phenylephrine (α1-adrenoceptor agonist; 1nM to 100µM), carbachol (muscarinic agonist; 1nM to 1mM) and α,β-methylene ATP (P2X receptor agonist; 1-10µM), as well as to electrical-field stimulation (EFS; 1-32Hz) were evaluated. PSM relaxations to isoproterenol (non-selective β-adrenoceptor agonist, 0.1nM to 10µM) and sodium nitroprusside (NO donor, 1nM to 10mM) were also evaluated. The ratio prostate weight/body weight was 22% greater (P<0.05) in L-NAME compared with control group. The PSM contractions to phenylephrine, carbachol and α,β-methylene ATP were higher in L-NAME (Emax: 3.85±0.25, 3.52±0.35 and 2.03±0.2mN, respectively) compared with control group (Emax: 3.08±0.17, 2.37±0.18 and 1.57±0.18mN, respectively). The PSM contractions induced by EFS were also significantly greater in L-NAME group. Prior incubation with the Rho-kinase inhibitor Y27632 (1µM) fully reversed the enhanced contractions to phenylephrine and carbachol. Isoproterenol-induced PSM relaxations were 34% lower in L-NAME group, which was associated with reduced levels of cAMP in prostate tissue. The relaxations to sodium nitroprusside remained unaltered in L-NAME group. In summary, chronic NO deficiency leads to increased Rho-kinase-mediated PSM contractile responses accompanied by impairment of β-adrenergic-cAMP-signaling pathway., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Association between hypertension and bladder symptoms has been described. We hypothesized that micturition dysfunction may be associated with renin-angiotensin system (RAS) acting in urethra. The effects of the anti-hypertensive drugs losartan (AT1 antagonist) and captopril (angiotensin-converting enzyme inhibitor) in comparison with atenolol (β1-adrenoceptor antagonist independently of RAS blockade) have been investigated in bladder and urethral dysfunctions during renovascular hypertension in rats. Two kidney-1 clip (2K-1C) rats were treated with losartan (30 mg/kg/day), captopril (50mg/kg/day) or atenolol (90 mg/kg/day) for eight weeks. Cystometric study, bladder and urethra smooth muscle reactivities, measurement of cAMP levels and p38 MAPK phosphorylation in urinary tract were determined. Losartan and captopril markedly reduced blood pressure in 2K-1C rats. The increases in non-voiding contractions, voiding frequency and bladder capacity in 2K-1C rats were prevented by treatments with both drugs. Likewise, losartan and captopril prevented the enhanced bladder contractions to electrical-field stimulation (EFS) and carbachol, along with the impaired relaxations to β-adrenergic-cAMP stimulation. Enhanced neurogenic contractions and impaired nitrergic relaxations were observed in urethra from 2K-1C rats. Angiotensin II also produced greater urethral contractions that were accompanied by higher phosphorylation of p38 MAPK in urethral tissues of 2K-1C rats. Losartan and captopril normalized the urethral dysfunctions in 2K-1C rats. In contrast, atenolol treatment largely reduced the blood pressure in 2K-1C rats but failed to affect the urinary tract smooth muscle dysfunction. The urinary tract smooth muscle dysfunction in 2K-1C rats takes place by local RAS activation irrespective of levels of arterial blood pressure., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The nitric oxide-cGMP signaling pathway modulates the ejaculatory functions. The nitric oxide (NO)-independent soluble guanylate cyclase haem-dependent stimulator BAY 41-2272 potently relaxes different types of smooth muscles. However, no study investigated its effects in vas deferens smooth muscle. Therefore, we designed experiments to evaluate the in vitro relaxing responses of vas deferens to BAY 41-2272. The effects of prolonged oral intake with BAY 41-2272 in vas deferens contractions of rats treated chronically with the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) were also investigated. BAY 41-2272 (0.001-100 μM) produced concentration-dependent relaxations in the prostatic and epididymal portions of vas deferens, an effect markedly reduced by the soluble guanylate cyclase inhibitor ODQ (100 μM). BAY 41-2272 significantly increased cGMP levels that were fully prevented by ODQ. In separate protocols, rats received L-NAME (20mg/rat/day) concomitantly with BAY 41-2272 (10mg/kg/day, 4 weeks), after which vas deferens contractions to electrical-field stimulation and noradrenaline were achieved. Electrical-field stimulation (1-32 Hz) evoked frequency-dependent contractions that were significantly enhanced in L-NAME-treated rats. Co-treatment with BAY 41-2272 fully reversed the increased contractile responses in L-NAME group. Noradrenaline (0.01-100 μM)-induced contractions were also greater in L-NAME-treated rats, and that was normalized by BAY 41-2272. In conclusion, BAY 41-2272 potently relaxes in vitro rat vas deferens smooth muscle and elevates the cGMP levels in an ODQ-sensitive manner. Moreover, prolonged oral intake with BAY 41-2272 restores the enhanced contractile vas deferens activity in rats treated with L-NAME. NO-independent soluble guanylate cyclase stimulators may be an alternative treatment for premature ejaculation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The soluble guanylyl cyclase is expressed in airway smooth muscle, and agents that stimulate this enzyme activity cause airway smooth muscle relaxation and bronchodilation. The compound 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent nitric oxide (NO)-independent soluble guanylyl cyclase stimulator, but little is known about its effects in airway smooth muscle. Therefore, this study aimed to investigate the mechanisms underlying the relaxations of rat tracheal smooth muscle induced by BAY 41-2272. Tracheal rings were mounted in 10-ml organ baths for isometric force recording. BAY 41-2272 concentration-dependently relaxed carbachol-precontracted tracheal rings (pEC(50)=6.68+/-0.14). Prior incubation with the NO synthesis inhibitor l-NAME (100 microM) or the soluble guanylyl cyclase inhibitor ODQ (10 microM) caused significant rightward shifts in the concentration-response curves to BAY 41-2272. Sodium nitroprusside caused concentration-dependent relaxations, which were greatly potentiated by BAY 41-2272 and completely inhibited by ODQ. In addition, BAY 41-2272 shifted to the right the tracheal contractile responses to either carbachol (0.01-1 microM) or electrical field stimulation (EFS, 1-32 Hz). BAY 41-2272 (1 microM) also caused a marked rightward shift and decreased the maximal contractile responses to extracellular CaCl2, and such effect was not modified by pretreatment with ODQ. In addition, BAY 41-2272 (up to 1 microM) significantly increased the cGMP levels, and that was abolished by ODQ. Our results indicate that BAY 41-2272 causes cGMP-dependent rat tracheal smooth muscle relaxations in a synergistic fashion with exogenous NO. BAY 41-2272 has also an additional mechanism independently of soluble guanylyl cyclase activation possibly involving Ca(2+) entry blockade., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The nitric oxide (NO)-independent soluble guanylyl cyclase stimulator stimulator BAY 41-2272 was reported to produce relaxant response in different types of smooth muscle. However no study was carried out to investigate the effects of BAY 412282 in detrusor smooth muscle. Thus, this study aimed to evaluate the relaxant effects of BAY 41-2272, in isolated mouse, rat and rabbit detrusor smooth muscle. Mouse, rat and rabbit were anesthetized, and urinary bladder removed. Detrusor smooth muscle was transferred to 10-mL organ baths containing oxygenated and warmed Krebs-Henseleit solution. Tissues were connected to force-displacement transducers and changes in isometric force were recorded. BAY 41-2272 (0.001-100 microM) produced concentration-dependent detrusor smooth muscle relaxations in mouse, rat and rabbit with maximal responses of 61.3+/-6.6%, 95.1+/-9.9% and 91.7+/-5.9%, respectively. Sodium nitroprusside and glyceryl trinitrate, as well as 8-bromo-cGMP also produced detrusor relaxations, but to a much lesser extent than BAY 41-2272. The NO synthesis inhibitor L-NAME and the phosphodiesterase-5 inhibitor sildenafil had no effect in BAY 41-2272-induced responses. However, the soluble guanylyl cyclase inhibitor ODQ significantly reduced BAY 41-2272-induced relaxations. BAY 41-2272 increased the bladder cGMP levels by about of 14- and 20-fold for 10 and 100 microM, respectively, which were markedly reduced by ODQ. The cAMP levels were unaffected by BAY 41-2272. Moreover, BAY 41-2272 significantly reduced the contractile responses to extracellular Ca(2+) in an ODQ-insensitive manner. In conclusion, rabbit detrusor smooth muscle relaxations by BAY 41-2272 involve mainly cGMP production, but an additional mechanism involving Ca(2+) influx blockade independently of cGMP production appears to be involved., (Copyright 2010 Elsevier B.V. All rights reserved.)