Basal release of 6-cyanodopamine from rat isolated vas deferens and its role on the tissue contractility.

6-Cyanodopamine is a novel catecholamine released from rabbit isolated heart. However, it is not known whether this catecholamine presents any biological activity. Here, it was evaluated whether 6-cyanodopamine (6-CYD) is released from rat vas deferens and its effect on this tissue contractility. Basal release of 6-CYD, 6-nitrodopamine (6-ND), 6-bromodopamine, 6-nitrodopa, and 6-nitroadrenaline from vas deferens were quantified by LC-MS/MS. Electric-field stimulation (EFS) and concentration-response curves to noradrenaline, adrenaline, and dopamine of the rat isolated epididymal vas deferens (RIEVD) were performed in the absence and presence of 6-CYD and /or 6-ND. Expression of tyrosine hydroxylase was assessed by immunohistochemistry. The rat isolated vas deferens released significant amounts of both 6-CYD and 6-ND. The voltage-gated sodium channel blocker tetrodotoxin had no effect on the release of 6-CYD, but it virtually abolished 6-ND release. 6-CYD alone exhibited a negligible RIEVD contractile activity; however, at 10 nM, 6-CYD significantly potentiated the noradrenaline- and EFS-induced RIEVD contractions, whereas at 10 and 100 nM, it also significantly potentiated the adrenaline- and dopamine-induced contractions. The potentiation of noradrenaline- and adrenaline-induced contractions by 6-CYD was unaffected by tetrodotoxin. Co-incubation of 6-CYD (100 pM) with 6-ND (10 pM) caused a significant leftward shift and increased the maximal contractile responses to noradrenaline, even in the presence of tetrodotoxin. Immunohistochemistry revealed the presence of tyrosine hydroxylase in both epithelial cell cytoplasm of the mucosae and nerve fibers of RIEVD. The identification of epithelium-derived 6-CYD and its remarkable synergism with catecholamines indicate that epithelial cells may regulate vas deferens smooth muscle contractility.

The multidrug resistance protein (MRP) 4 is expressed and functionally active in isolated bladder from pig.

Multidrug resistance proteins type 4 (MRP4) and 5 (MRP5) play pivotal roles in the transport of cyclic nucleotides in various tissues. However, their specific functions within the lower urinary tract remain relatively unexplored. This study aimed to investigate the effect of pharmacological inhibition of MRPs on cyclic nucleotide signaling in isolated pig bladder. The relaxation responses of the bladder were assessed in the presence of the MRP inhibitor, MK571. The temporal changes in intra- and extracellular levels of cAMP and cGMP in stimulated tissues were determined by mass spectrometry. The gene (ABCC4) and protein (MRP4) expression were also determined. MK571 administration resulted in a modest relaxation effect of approximately 26% in carbachol-pre-contracted bladders. The relaxation induced by phosphodiesterase inhibitors such as cilostazol, tadalafil, and sildenafil was significantly potentiated in the presence of MK571. In contrast, no significant potentiation was observed in the relaxation induced by substances elevating cAMP levels or stimulators of soluble guanylate cyclase. Following forskolin stimulation, both intracellular and extracellular cAMP concentrations increased by approximately 15.8-fold and 12-fold, respectively. Similarly, stimulation with tadalafil + BAY 41-2272 resulted in roughly 8.2-fold and 3.4-fold increases in intracellular and extracellular cGMP concentrations, respectively. The presence of MK571 reduced only the extracellular levels of cGMP. This study reveals the presence and function of MRP4 transporters within the porcine bladder and paves the way for future research exploring the role of this transporter in both underactive and overactive bladder disorders.

The pivotal role of neuronal nitric oxide synthase in the release of 6-nitrodopamine from mouse isolated vas deferens.

6-Nitrodopamine (6-ND) is released from rat and human vas deferens and is considered a major mediator of both tissues contractility. The contractions induced by 6-ND are selectively blocked by both tricyclic antidepressants and α1-adrenoceptor antagonists. Endothelial nitric oxide synthase (eNOS) is the major isoform responsible for 6-ND release in mouse isolated heart, however the origin of 6-ND in the vas deferens is unknown. Here it was investigated by LC-MS/MS the basal release of 6-ND from isolated vas deferens obtained from control, eNOS-/-, nNOS-/-, and iNOS-/- mice. In addition, it was evaluated in vitro vas deferens contractility following electric field stimulation (EFS). Basal release of 6-ND was significantly reduced in nNOS-/- mice compared to control mice, but not decreased when the vas deferens were obtained from either eNOS-/- or iNOS-/- mice. Pre-incubation of the vas deferens with tetrodotoxin (1 µM) significantly reduced the basal release of 6-ND from control, eNOS-/-, and iNOS-/- mice but had no effect on the basal release of 6-ND from nNOS-/- mice. EFS-induced frequency-dependent contractions of the vas deferens, which were significantly reduced when the tissues obtained from control, eNOS-/- and iNOS-/- mice, were pre-incubated with l-NAME, but unaltered when the vas deferens was obtained from nNOS-/- mice. In addition, the EFS-induced contractions were significantly smaller when the vas deferens were obtained from nNOS-/- mice. The results clearly demonstrate that nNOS is the main NO isoform responsible for 6-ND release in mouse vas deferens and reinforces the concept of 6-ND as a major modulator of vas deferens contractility.

A 2-week treatment with 5-azacytidine improved the hypercontractility state in prostate from obese mice: Role of the nitric oxide-cyclic guanosine monophosphate signalling pathway.

Benign prostatic hyperplasia (BPH) is characterised by increases in prostate volume and contraction. Downregulation of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway contributes to prostate dysfunctions. Previous studies in cancer cells or vessels have shown that the epigenetic mechanisms control the gene and protein expression of the enzymes involved in the production of NO and cGMP. This study is aimed to evaluate the effect of a 2-week treatment of 5-azacytidine (5-AZA), a DNA-methyltransferase inhibitor, in the prostate function of mice fed with a high-fat diet. Functional, histological, biochemical and molecular assays were carried out. Obese mice presented greater prostate weight, α-actin expression and contractile response induced by the α-1adrenoceptors agonist. The relaxation induced by the NO-donor and the protein expression of endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) were significantly decreased in the prostate of obese mice. The treatment with 5-AZA reverted the higher expression of α-actin, reduced the hypercontractility state of the prostate and increased the expression of eNOS and sGC and intraprostatic levels of cGMP. When prostates from obese mice treated with 5-AZA were incubated in vitro with inhibitors of the NOS or sGC, the inhibitory effect of 5-AZA was reverted, therefore, showing the involvement of NO and cGMP. In conclusion, our study paves the way to develop or repurpose therapies that recover the expression of eNOS and sGC and, hence, to improve prostate function in BPH.

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.

Metformin Counteracts the Deleterious Effects of Methylglyoxal on Ovalbumin-Induced Airway Eosinophilic Inflammation and Remodeling.

Exposure to methylglyoxal (MGO) increases the levels of receptor for advanced glycation end products (RAGE) and reactive-oxygen species (ROS) in mouse airways, exacerbating the inflammatory responses. Metformin scavenges MGO in plasma of diabetic individuals. We investigated if amelioration by metformin of eosinophilic inflammation reflects its ability to inactivate MGO. Male mice received 0.5% MGO for 12 weeks together or not with 2-week treatment with metformin. Inflammatory and remodeling markers were evaluated in bronchoalveolar lavage fluid (BALF) and/or lung tissues of ovalbumin (OVA)-challenged mice. MGO intake elevated serum MGO levels and MGO immunostaining in airways, which were reduced by metformin. The infiltration of inflammatory cells and eosinophils and levels of IL-4, IL-5 and eotaxin significantly increased in BALF and/or lung sections of MGO-exposed mice, which were reversed by metformin. The increased mucus production and collagen deposition by MGO exposure were also significantly decreased by metformin. In MGO group, the increases of RAGE and ROS levels were fully counteracted by metformin. Superoxide anion (SOD) expression was enhanced by metformin. In conclusion, metformin counteracts OVA-induced airway eosinophilic inflammation and remodeling, and suppresses the RAGE-ROS activation. Metformin may be an option of adjuvant therapy to improve asthma in individuals with high levels of MGO.

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.

Urethral dysfunction in a rat model of chemically induced prostatic inflammation: potential involvement of the MRP5 pump.

Prostate inflammation (PI) is a clinical condition associated with infection and/or inflammation of the prostate. It is a common disease frequently associated to lower urinary tract (LUT) symptoms. The urethra is an understudied structure in the LUT and plays a fundamental role in the urinary cycle. Here, we proposed to evaluate the effect of PI on the urethra tissue. Male Sprague-Dawley rats were used, and PI was induced by formalin injection into the ventral lobes of the prostate. The pelvic urethra at the prostatic level was harvested for histological analysis, contraction (electrical field stimulation and phenylephrine), and relaxation (sodium nitroprusside/MK-571) experiments. Various gene targets [cytochrome c oxidase subunit 2, transforming growth factor-ß1, interleukin-1ß, hypoxia-inducible factor-1α, α1A-adrenoceptor, inositol 1,4,5-trisphosphate receptor type 1, voltage-gated Ca2+ channel subunit-α1D, neuronal nitric oxide synthase, soluble guanylyl cyclase, phosphodiesterase 5A, protein kinase CGMP-dependent 1, and multidrug resistance-associated protein 5 (MRP5; ATP-binding cassette subfamily C member 5)] were quantified, and cGMP levels were measured. No histological changes were detected, and functional assays revealed decreased contraction and increased relaxation of urethras from the PI group. The addition of MK-571 to functional assays increased urethral relaxation. Genes associated with inflammation were upregulated in urethras from the PI group, such as cytochrome oxidase c subunit 2, transforming growth factor-ß1, interleukin-1ß, and hypoxia-inducible factor-1α. We also found increased expression of L-type Ca2+ channels and the neuronal nitric oxide synthase enzyme and decreased expression of the MRP5 pump. Finally, cGMP production was enhanced in urethral tissue of PI animals. The results indicate that PI is associated with proinflammatory gene expression in the urethra without histologically evident inflammation and that PI produces a dysfunctional urethra and MRP5 pump downregulation, which results in cGMP accumulation inside the cell. These findings would help to better understand LUT dysfunctions associated with PI and the role of MRP pumps in the control of LUT function.

Lorcaserin Administration has Pro-Ejaculatory Effects in Rats via 5-HT2C Receptors Activation: A Putative Pharmacologic Strategy to Delayed Ejaculation?

BACKGROUND: Lorcaserin is an anti-obesity drug whose weight loss effect results from 5-hydroxytryptamin (5-HT)2C receptors activation. The 5-HT2C receptor was shown to participate in the physiological control of ejaculation, but no data addressing a putative effect of lorcaserin on ejaculation exist. AIM: To investigate the effects of lorcaserin in different in vitro and in vivo experimental models of ejaculation in rats. METHODS: Contractile responses to lorcaserin in rat seminal emission organs in vitro (prostatic and epididymal vas deferens, cauda epididymis, and seminal vesicles), analysis of male rat copulatory behavior, and electromyographic recording of bulbospongiosus muscle in anesthetized animals were studied. MAIN OUTCOME MEASURES: The main outcome measures included in vitro contraction of seminal emission organs and evaluation of the male rat copulatory behavior. The male rat sexual behavior in terms of copulation latency, ejaculation latency, mount and intromission frequency, and ejaculation frequency of sexually experienced adult male rats with a receptive female were also recorded. RESULTS: Lorcaserin (1.0 nM to 1.0 mM) had no significant effects on the in vitro contractility of seminal emission organs smooth muscle (cauda epididymis, vas deferens, and seminal vesicles). On the other hand, lorcaserin administration (0.3-1.0 mg/kg, intravenous) induced ejaculation in anesthetized rats, which was prevented by the 5-HT2C-selective antagonist SB 242084 (0.1 and 0.3 mg/kg, intravenous). Single-dose treatment of non-anesthetized male rats with lorcaserin (1.0, 4.0, or 10 mg/kg, per os) induced non-copulating ejaculations in sexually naïve rats. Lorcaserin also had pro-ejaculation effects by decreasing the ejaculation threshold of copulating rats by half. The pro-ejaculatory effects of lorcaserin were reversible as the ejaculation threshold of treated rats recovered after a 1-week washout period. CLINICAL IMPLICATIONS: Due to its reported clinical safety, repurposing lorcaserin for the treatment of delayed ejaculation may be suggested. STRENGTHS & LIMITATIONS: The pro-ejaculatory effect of lorcaserin administration and the role of 5-HT2C were demonstrated in different experimental models of ejaculation in rats. The lack of studies in putative experimental models of delayed ejaculation is a limitation of this study. CONCLUSION: Our results demonstrate that the clinically approved 5-HT2C agonist lorcaserin is a strong facilitator of ejaculation in rats. de Almeida Kiguti LR, Pacheco TL, Antunes E, et al. Lorcaserin Administration has Pro-Ejaculatory Effects in Rats via 5-HT2C Receptors Activation: A Putative Pharmacologic Strategy to Delayed Ejaculation? J Sex Med 2020;17:1060-1071.

Guanosine, a guanine-based nucleoside relaxed isolated corpus cavernosum from mice through cGMP accumulation.

In corpus cavernosum (CC), guanosine triphosphate (GTP) is converted into cyclic guanosine monophosphate (cGMP) to induce erection. The action of cGMP is terminated by phosphodiesterases and efflux transporters, which pump cGMP out of the cell. The nucleotides, GTP, and cGMP were detected in the extracellular space, and their hydrolysis lead to the formation of intermediate products, among them guanosine. Therefore, our study aims to pharmacologically characterize the effect of guanosine in isolated CC from mice. The penis was isolated and functional and biochemical analyses were carried out. The guanine-based nucleotides GTP, guanosine diphosphate, guanosine monophosphate, and cGMP relaxed mice corpus cavernosum, but the relaxation (90.7 ± 12.5%) induced by guanosine (0.000001-1 mM) was greater than that of the nucleotides (~ 45%, P < 0.05). Guanosine-induced relaxation was not altered in the presence of adenosine type 2A and 2B receptor antagonists. No augment was observed in the intracellular levels of cyclic adenosine monophosphate in tissues stimulated with guanosine. Inhibitors of nitric oxide synthase (L-NAME, 100 µM) and soluble guanylate cyclase (ODQ, 10 µM) produced a significant reduction in guanosine-induced relaxation in all concentrations studied, while in the presence of tadalafil (300 nM), a significant increase was observed. Pre-incubation of guanosine (100 µM) produced a 6.6-leftward shift in tadalafil-induced relaxation. The intracellular levels of cGMP were greater when CC was stimulated with guanosine. Inhibitors of ecto-nucleotidases and xanthine oxidase did not interfere in the response induced by guanosine. In conclusion, our study shows that guanosine relaxes mice CC and opens the possibility to test its role in models of erectile dysfunction.

Neurotoxicity of Tityus bahiensis (brown scorpion) venom in sympathetic vas deferens preparations and neuronal cells.

Systemic scorpion envenomation is characterized by massive neurotransmitter release from peripheral nerves mediated primarily by scorpion venoms neurotoxins. Tityus bahiensis is one of the medically most important species in Brazil, but its venom pharmacology, especially regarding to peripheral nervous system, is poorly understood. Here, we evaluated the T. bahiensis venom activity on autonomic (sympathetic) neurotransmission by using a variety of approaches, including vas deferens twitch-tension recordings, electrophysiological measurements (resting membrane potentials, spontaneous excitatory junctional potentials and whole-cell patch-clamp), calcium imaging and histomorphological analysis. Low concentrations of venom (≤ 3 µg/mL) facilitated the electrically stimulated vas deferens contractions without affecting postsynaptic receptors or damaging the smooth muscle cells. Transient TTX-sensitive sustained contractions and resting membrane depolarization were mediated mainly by massive spontaneous ATP release. High venom concentrations (≥ 10 µg/mL) blocked the muscle contractions and induced membrane depolarization. In neuronal cells (ND7-23wt), the venom increased the peak sodium current, modified the current-voltage relationship by left-shifting the Nav-channel activation curve, thereby facilitating the opening of these channels. The venom also caused a time-dependent increase in neuronal calcium influx. These results indicate that the sympathetic hyperstimulation observed in systemic envenomation is presynaptically driven, probably through the interaction of α- and ß-toxins with neuronal sodium channels.

Amiloride Relaxes Rat Corpus Cavernosum Relaxation In Vitro and Increases Intracavernous Pressure In Vivo.

INTRODUCTION: The antihypertensive effects of thiazide diuretics such as hydrochlorothiazide are commonly associated with erectile dysfunction. The association of hydrochlorothiazide/amiloride is not associated with erectile dysfunction. The hypothesis is that amiloride has beneficial effect in penile erection and, therefore, counterbalances the hydrochlorothiazide-induced disruptive effect. AIM: To investigate the effects of amiloride and its analogues hexamethylamiloride and benzamil on rat isolated corpus cavernosa (CC) and intracavernous pressure (ICP) in anaesthetized rats. METHODS: Rat isolated CC were incubated with amiloride, hexamethylamiloride, and benzamil (10 and 100 µmol/L each), followed by phenylephrine, potassium chloride, and electrical field stimulation (EFS). Their effect on the relaxant responses to EFS and sodium nitroprusside were also determined. Oral (30 mg/kg) and intraperitoneal (3 mg/kg) treatments with amiloride were also investigated on nerve-evoked ICP. MAIN OUTCOME MEASURES: In vitro functional studies and in vivo ICP measurement on rat CC were performed. Additionally, phosphodiesterase type V isoform A1 activity and the mRNA expressions of Na+/H+ pump, epithelial sodium channel exchangers (ENaC) channels (α-, ß- and γ subunits) and Na+/Ca2+ exchangers were evaluated in CC tissues. RESULTS: Amiloride and its analogues significantly reduced the phenylephrine-, potassium chloride-, and EFS-induced CC contractions, which were not changed by nitro-L-arginine methyl ester (100 µmol/L) or indomethacin (6 µmol/L). In phenylephrine-precontracted CC tissues, amiloride itself caused concentration-dependent relaxation and significantly increased the EFS-induced relaxation. Oral and intraperitoneal treatment with amiloride significantly increased the ICP. Phosphodiesterase type V isoform A1 activity was not affected by amiloride. Na+/H+ pump, ENaC, and Na+/Ca2+ exchanger mRNA expressions were all detected in rat CC tissues. CLINICAL IMPLICATION: Amiloride analogues may have therapeutic potential for erectile dysfunction. STRENGTH & LIMITATIONS: The interesting effect of amiloride in penile erection was observed in both in vitro and in vivo methods. The evidence at the moment is restricted to rat CC. CONCLUSION: Amiloride reduces in vitro CC contractility and enhances erectile function after oral and intraperitoneal administration, possibly via inhibition of ENaC. Campos R, Claudino MA, de Oliveira MG, et al. Amiloride Relaxes Rat Corpus Cavernosum Relaxation In Vitro and Increases Intracavernous Pressure In Vivo. J Sex Med 2019;16:500-511.

Autonomic dysregulation at multiple sites is implicated in age-associated underactive bladder in female mice.

AIMS: To evaluate the functional and molecular alterations of contractile and relaxant machinery in the bladder and urethra that lead to the underactive bladder (UAB) in old female mice. METHODS: Female young (3-months) and old (18-months) C57BL/6 mice were used. Urodynamic was assessed in awake and anaesthetized mice. Electrical-field stimulation (EFS) and concentration-response curves to contractile and relaxing agents in isolated bladders and urethras were performed. Messenger RNA (mRNA) expressions of muscarinic, adrenergic, and transient receptor potential vanilloid-4 (TRPV4), and of the enzymes tyrosine hydroxylase and neuronal nitric oxide synthase (nNOS) were determined. Bladder cyclic adenosine monophosphate (cAMP) levels were measured. RESULTS: Cystometry in old mice showed incapacity to produce bladder emptying. On filter paper, old mice showed reduced urinary spots. Compared to the young group, bladder contractions induced by EFS and carbachol were lower in old mice. The ß3 -adrenoceptor agonist mirabegron promoted higher bladder relaxation and elevation of cAMP levels in old mice. In old mice urethras, the α1a -adrenoceptor agonist phenylephrine produced higher contractions, but no differences were found for the NO donor sodium nitroprusside-induced relaxations. In old mice, increased mRNA expressions of ß3 - and α1a -adrenoceptors in bladder and urethra were found, respectively, whereas the muscarinic M2 and M3 receptors and ß2 -adrenoceptors did not change between groups. Reduced mRNA expressions of tyrosine hydroxylase and nNOS were found in old mouse urethras. Additionally, TRPV4 expression was reduced in bladder urothelium from old mice. CONCLUSION: Age-associated mouse UAB is the result of autonomic dysfunction at multiple levels leading to the less sensitive and overrelaxed bladder, along with urethral hypercontractility.

Tumor necrosis factor alpha has a crucial role in increased reactive oxygen species production in platelets of mice injected with lipopolysaccharide.

Increased reactive oxygen species (ROS) production leads to tissue damage observed in sepsis and lipopolysaccharide (LPS)-exposed animals. LPS stimulates cytokines releasing, including tumor necrosis factor alpha (TNF-α), that is important to ROS production. Platelets, considered inflammatory cells, generate ROS when exposed to LPS in vivo, but not when they are incubated in vitro with this compound. Therefore, we investigated the role of TNF-α on the increased intraplatelet ROS levels after LPS treatment. Mice were injected with LPS (1 mg/kg) or TNF-α (10 ng/kg), and blood was collected to prepare the washed platelets. Animals were treated with infliximab (anti-TNF-α antibody), R-7050 (non-selective TNF-α receptor antagonist) or apocynin (NADPH oxidase inhibitor). At 48 h after LPS or TNF-α injection, the ROS levels in ADP (25 µM)-activated platelets were evaluated by flow cytometry. Our data showed that injection of mice with LPS increased by 4-fold the ROS production (p < 0.05), which was significantly reduced by the treatments with infliximab, R-7050 or apocynin. Injection of mice with TNF-α markedly elevated the ROS formation in platelets (p < 0.05) that was reduced by infliximab, R-7050 or apocynin treatments. In separate experiments, platelets from saline-injected mice were incubated with TNF-α (30 to 3000 pg/mL) in absence or presence of infliximab, R-7050, apocynin or GKT137831 (NOX1/NOX4 inhibitor) before ROS measurements. TNF-α in vitro markedly increased the ROS levels, an effect significantly reduced by all treatments. Therefore, platelets are involved in the oxidative stress induced by LPS through TNF-α action, and NADPH oxidase takes part in this effect.

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.

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.

Inhibition of Multidrug Resistance Proteins by MK 571 Enhances Bladder, Prostate, and Urethra Relaxation through cAMP or cGMP Accumulation.

The biologic effect of cAMP and cGMP is terminated by phosphodiesterases and multidrug resistance proteins MRP4 and MRP5, which pump cyclic nucleotides out of the cell. Therefore, this study aimed to characterize the role of MRP inhibitor, MK 571 (3-[[[3-[(1E)-2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propanoic acid), in the bladder, prostate, and urethra of male mice by means of functional assays, protein expression, and cyclic nucleotide quantification. The cumulative addition of MK 571 (1-30 µM) produced only small relaxation responses (approximately 25%) in all studied tissues. In the bladder, isoprenaline/fenoterol and forskolin concentration-dependently relaxed and MK 571 (20 µM) increased the maximal response values by 37% and 24%, respectively. When MK 571 was coincubated with fenoterol or forskolin, intracellular levels of cAMP and protein expression of phospho-vasodilator-stimulated phosphoprotein (p-VASP) Ser157 were significantly greater compared with bladders stimulated with fenoterol or forskolin alone. In the prostate and urethra, sodium nitroprusside concentration-dependently relaxed and MK 571 (20 µM) significantly increased relaxation responses by 70% and 56%, respectively, accompanied by greater intracellular levels of cGMP and protein expression of p-VASP Ser239 in the prostate. Tadalafil and BAY 41-2272 (5-cyclopropyl-2-[1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl]-4-pyrimidinamine) also relaxed the prostate and urethra, respectively, and MK 571 markedly enhanced this response. The stable analog of cGMP (8-Br-cGMP) induced concentration-dependent relaxation responses in the prostate and urethra, and MK 571 significantly increased the relaxation response. In conclusion, to our knowledge, this is the first study to show that efflux transporters are physiologically active in the bladder, prostate, and urethra to control intracellular levels of cAMP or cGMP.

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.

How important is the α1-adrenoceptor in primate and rodent proximal urethra? Sex differences in the contribution of α1-adrenoceptor to urethral contractility.

Urethral smooth muscle (USM) contributes to urinary continence by contracting during the urine storage phase, which is mainly mediated by activation of postjunctional α1-adrenoceptors. Males and females show differences in the functioning of the lower urinary tract and the most common urinary tract symptoms (LUTS). LUTS in men typically occur in association with bladder outlet obstruction, whereas in women urinary urge-incontinence symptoms are more common. Therefore, this study aimed to evaluate sex differences in α1-adrenoceptor subtype expression and their importance in proximal urethra contraction in the mouse (C57BL6/J) and marmoset (Callithrix jacchus). Contractile responses to phenylephrine, norepinephrine, potassium chloride (KCl), and electrical-field stimulation (EFS) were evaluated. Phenylephrine, norepinephrine, KCl, and EFS produced markedly greater contractions in male mice and marmoset USM compared with females. The sex differences remained unchanged by Nω-nitro-l-arginine (l-NAME; nitric oxide synthase inhibitor), atropine (muscarinic receptor antagonist), and PPADS (P2X1-purinoceptor antagonist). Additionally, selective α1A (but not α1B- and α1D-)-adrenoceptor antagonists significantly reduced phenylephrine-induced USM contractions. qRT-PCR for α1A-, B-, and D-adrenoceptor subtypes revealed a marked presence of the α1A-adrenoceptor subtype in male USM, but not females. Male mouse urethra also exhibited a higher tyrosine hydroxylase mRNA expression. Histomorphometric analysis showed a greater USM area in male than female mice. In conclusion, male mouse and marmoset proximal USM shows strong α1A- adrenoceptor-induced contractions and abundant α1A-adrenoceptor expression, whereas α1A-adrenoceptor-mediated mechanisms are much less important in females. The differential expression of α1-adrenoceptors in the proximal urethra may contribute to the higher incidence of urinary incontinence in women and obstructed voiding in men.