Detrusor Smooth Muscle Contraction Research Articles

PTHrP attenuates spontaneous contractions in detrusor smooth muscle of the rat bladder by activating spontaneous transient outward potassium currents.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) cells upon bladder distension attenuates spontaneous phasic contractions (SPCs) in DSM and associated afferent firing to facilitate urine storage. Here, we investigate the mechanisms underlying PTHrP-induced inhibition of SPCs, focusing on large-conductance Ca2+-activated K+ channels (BK channels) that play a central role in stabilizing DSM excitability. Perforated patch-clamp techniques were applied to DSM cells of the rat bladder dispersed using collagenase. Isometric tension changes were recorded from DSM strips, while intracellular Ca2+ dynamics were visualized using Cal520AM-loaded DSM bundles. DSM cells developed spontaneous transient outward potassium currents (STOCs) arising from the opening of BK channels. PTHrP (10nM) increased the frequency of STOCs without affecting their amplitude at a holding potential of - 30mV but not - 40mV. PTHrP enlarged depolarization-induced, BK-mediated outward currents at membrane potentials positive to + 20mV in a manner sensitive to iberiotoxin (100nM), the BK channel blocker. The PTHrP-induced increases in BK currents were also prevented by inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (CPA 10µM), L-type voltage-dependent Ca2+ channel (LVDCC) (nifedipine 3µM) or adenylyl cyclase (SQ22536 100µM). PTHrP had no effect on depolarization-induced LVDCC currents. PTHrP suppressed and slowed SPCs in an iberiotoxin (100nM)-sensitive manner. PTHrP also reduced the number of Ca2+ spikes during each burst of spontaneous Ca2+ transients. In conclusion, PTHrP accelerates STOCs discharge presumably by facilitating SR Ca2+ release which prematurely terminates Ca2+ transient bursts resulting in the attenuation of SPCs.

B-308 Focal Adhesion Kinase Activation is Involved in Tension Development During Contractile Stimulation of Mouse Detrusor Smooth Muscle to Modulate Bladder Dysfunction-Related Lower Urinary Tract Symptoms

Abstract Background The function of the bladder is to store and expel urine. Bladder dysfunction-related lower urinary tract symptoms (LUTS) is a common urological problem and negatively affects health-related quality of life. Investigation of detrusor muscle contraction mechanisms is necessary to understand its underlying pathophysiology. Voiding occurs through bladder muscle contraction, which is mediated primarily by the increased intracellular Ca2+ concentration-induced myosin regulatory light chain (MLC) phosphorylation. Recent studies showed that focal adhesion kinase (FAK)-regulated focal adhesion assembly and actin polymerization are involved in vascular and airway smooth muscle contraction. However, the role of FAK activation in visceral smooth muscle contraction is not well documented. This study aims to determine if FAK activation is involved in mouse detrusor smooth muscle contraction. Methods Normal adult male and female mouse bladders were harvested. The trigone and dome areas were cut, and the bladder body was used. The mucosa was carefully removed. Detrusor muscle strips (1.5–2 × 8–10 mm) were prepared and then suspended in a 20 mL tissue bath filled with physiological Krebs solution and bubbled with 95% O2 and 5% CO2 to obtain a pH of 7.4 at 37 °C. The isometric contraction was recorded. After establishing a stable baseline, the strips were stimulated with 120 mM potassium chloride (KCl), which was used to normalize the contractility induced by subsequent stimuli. One of the following specific inhibitors or the same volume of vehicle (DMSO) was applied and incubated for 30 min: PF-573228 (2 µM, a FAK inhibitor) or latrunculin B (1 µM, an inhibitor of actin polymerization). Then, the contractile responses to KCl (90 mM), electrical field stimulation (EFS, 2–32 Hz), or carbachol (CCh, 10−7.5–10−4.5 M ) were measured. In a separate experiment, the detrusor strips were stimulated with CCh (10 µM) after incubation with PF-573228 (2 µM) or vehicle (DMSO) for 30 min. Once the peak contraction was reached, the strips were snap-frozen for immunoblotting analysis of the phosphorylated FAK (p-FAK) and MLC (p-MLC). Statistical analysis was performed (GraphPad Prism 6). The contractile responses to KCl and EFS between vehicle and inhibitor groups were compared using a paired t-test. Nonlinear regression was used to compare CCh-induced dose-response curves. For the expression of phosphorylated proteins, one-way ANOVA, followed by Tukey’s posthoc test, was used for analysis. Results KCl-induced contractile responses decreased significantly after incubation with PF-573228 or latrunculin B compared to the corresponding vehicle group (P < 0.0001). The contractile responses induced by EFS were significantly inhibited by preincubation with PF-573228 at 8, 16, and 32 Hz (P < 0.05) or latrunculin B at 16 and 32 Hz (P < 0.01). Following the application of PF-573228 or latrunculin B, CCh-induced dose-response contractions were significantly lower than those in the corresponding vehicle-treated strips (P = 0.0021 and 0.0003, respectively). Immunoblotting analysis demonstrated that CCh stimulation caused increased expression of p-FAK and p-MLC, whereas preincubation with PF-573228 led to decreased expression of p-FAK but not p-MLC Conclusion FAK activation is involved in tension development induced by contractile stimulation in mouse detrusor muscle, probably through enhancing actin polymerization, but not MLC phosphorylation.

MP03-07 THE LACK OF MECHANOSENSITIVE TREK-1 CHANNEL MIMICS THE DEVELOPMENT OF AGING-RELATED BLADDER PHENOTYPES IN MICE

You have accessJournal of UrologyCME1 Apr 2023MP03-07 THE LACK OF MECHANOSENSITIVE TREK-1 CHANNEL MIMICS THE DEVELOPMENT OF AGING-RELATED BLADDER PHENOTYPES IN MICE Alison Xiaoqiao Xie, Nao Iguchi, and Anna Malykhina Alison Xiaoqiao XieAlison Xiaoqiao Xie More articles by this author , Nao IguchiNao Iguchi More articles by this author , and Anna MalykhinaAnna Malykhina More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003214.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Previous animal and human studies have confirmed that the potassium channel subfamily K member 2 (TREK-1), a member of two-pore domain mechanosensitive K+ channels (K2P), acts as a mechanosensor in the urinary bladder. We reported that TREK-1 is a predominantly expressed K2P channel in the human detrusor, and its expression and function are altered in patients with idiopathic detrusor overactivity (DO). TREK-1 expression in the peripheral organs and in the nervous system also declines during aging. This study aimed to investigate the role of TREK-1-related mechanosensitivity in the detrusor in aging mice. METHODS: Bladder histology, detrusor contractility, and voiding function were investigated in adult (3, 6, 9 and 12 months old) TREK-1 KO mice of both sexes and age-matched wildtype controls. Bladder weight, detrusor smooth muscle (DSM) thickness, and collagen content were compared between the groups using paraffin-embedded bladder samples. Bladder strip assay was used to evaluate detrusor muscle contractility and nerve-induced bladder contractions. Voiding spot assay (VSA) and awake cystometry recordings were performed to assess micturition patterns in vivo. RESULTS: Increased bladder weight and bladder wall thickness were observed in TREK-1 KO mice of both sexes, starting as early as at 6-month-old. and threshold volume in cystometry recordings. In comparison to age- and sex-matched controls, bladder strips isolated from TREK-1 KO mice showed significantly decreased DSM contractility including contractile responses to electric field stimulation and cholinergic receptor activation. Interestingly, while the peak forces of evoked DSM contractions decreased with aging in control mice, TREK-1 KO animals at 6-12 month-old displayed DSM contractility that was lower than in 12-month-old control animals. Increased numbers of non-voiding contractions were observed in both sexes of TREK-1 KO mice, whereas the bladder capacity, intermicturition interval, and micturition threshold volume were significantly increased in comparison to age-matched controls. CONCLUSIONS: In summary, the lack of TREK-1 expression in adult mice significantly increased a number of functional changes typically observed in underactive bladders with detrusor overactivity, including increases in bladder size, weight, capacity, and a significant decrease in DSM contractility. Our findings suggest that decreased TREK-1 expression may mimic the development of age-related voiding phenotypes in mice. Source of Funding: DK095817 (Malykhina), DK116648 (Malykhina) and Department of Surgery Academic Enrichment Fund (Xie) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e23 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alison Xiaoqiao Xie More articles by this author Nao Iguchi More articles by this author Anna Malykhina More articles by this author Expand All Advertisement PDF downloadLoading ...

Sodium Nitroprusside Inhibits Detrusor Muscle Contraction through G-protein Mediated Inhibition of Intracellular Ca2+ Release

Background: Nitric oxide (NO) physiology is very complex in detrusor smooth muscle (DSM). While NO relaxes detrusor in some species, in contrary, it produces contraction in other. NO donor sodium nitroprusside (SNP) was found to inhibit CCh-induced contraction in goat DSM in earlier studies, but it is not known how SNP produces inhibitory action on goat DSM. Therefore, the aim of this in vitro study was to investigate the mechanism of inhibitory action of SNP pertaining to involvement of G- protein and other second messenger on goat DSM. Methods: Goat detrusor muscle strips collected from local abattoir were mounted in a thermostatically controlled (37o±0.5oC) organ bath (20 ml capacity) containing physiological solution. Following 1 hr of equilibration, carbachol (CCh) (10-5 M) induced sub-maximal contraction was elicited both in the absence and presence of SNP (10-5 M). Involvement of NO and guanylyl cyclase was assessed using nitric oxide synthase inhibitor L-NAME (10-5 M) and guanylyl cyclase inhibitor ODQ (10-5 M). Experiments were also carried out in the presence of G-protein activator aluminium fluoride (AlF) (10-5 M), protein kinase C (PKC) activator phorbol-12-myristate (PMA) (10-5 M), nifedipine, low Ca2+ PSS, theophylline and Zero Ca2+ PSS. Result: Inhibitory effect of SNP (10-5 M) on CCh-induced contraction on goat DSM was reversed by nitric oxide synthase inhibitor L-name (10-5 M) and guanylyl cyclase inhibitor ODQ (10-5 M). Prior incubation of the tissues with AlF and PMA also reversed the SNP mediated inhibition. On the other hand, nifedipine and low Ca2+ PSS; and theophylline and zero Ca2+ PSS potentiated the SNP-mediated inhibition. Thus, the present study shows that SNP elicited inhibitory effect on goat DSM is both NO-dependent and cGMP-dependent. In addition, the result shows that SNP inhibits G-protein coupled PKC leading to inhibition of both Ca2+ entry and intracellular Ca2+ release with a resultant decrease in intracellular Ca2+.

Lys5,MeLeu9,Nle10]-NKA(4-10) induces neurokinin 2 receptor mediated urination and defecation and neurokinin 1 receptor mediated flushing in rats: measured using the rapid detection voiding assay.

Neurokinin 2 receptor (NK2R) agonists may be useful for treating bladder and bowel dysfunction via direct contraction of detrusor and gastrointestinal smooth muscle. The NK2R agonist [Lys5, MeLeu9, Nle10]-NKA(4-10) (LMN-NKA) induces urination and defecation, but also produces the potential side effect of dermal flushing in rats. Although LMN-NKA is a NK2R agonist, italso has affinity for neurokinin 1 receptors (NK1R). Therefore, the goal of this study was to determine the neurokinin receptor (NKR) subtypes responsible for LMN-NKA-induced urination, defecation, and flushing byblocking either NK2Rs or NK1Rs before LMN-NKA administration. To accomplish this goal, we developed a simple high-throughput 'rapid detection voiding assay' to detect rapid-onset drug-induced urination and defecation in rats. In LMN-NKA dose-response experiments, LMN-NKA (10-100μg/kg, subcutaneous) was injected and urination, defecation, and flushing were monitored for 30min. For NKR antagonist experiments, vehicle, the NK2R antagonist GR159897, or the NK1R antagonist CP-99,994 were injected before an acclimation period. Following acclimation, saline or 100μg/kg LMN-NKA were injected, and behavior was observed for 30min. LMN-NKA produced dose-related increases in urination, defecation, and flushing. Blocking NK2Rs reduced urination and blocked defecation, without affecting flushing. Blocking NK1Rs did not change LMN-NKA-induced urination or defecation but reduced LMN-NKA-induced flushing. Using the rapid detection voiding assay we show that LMN-NKA-induced urination and defecation are mediated by NK2Rs, while flushing is mediated by NK1Rs. Therefore, drugs that are more selective for NK2 vs. NK1Rs should produce rapid-onset urination and defecation without producing the potential side effect of flushing.

Selective reduction of neurotransmitter release by cAMP-dependent pathways in mouse detrusor.

Parasympathetic nerve-mediated contractions of detrusor smooth muscle are generated by ATP and acetylcholine (ACh) release from efferent nerve terminals. In humans, ACh is responsible for detrusor contractions in normal human bladders, whereas ATP has an additional role in overactive bladder pathologies. The ATP metabolite, adenosine, relaxes nerve-mediated contractions, with a potential action via presynaptic adenosine A1 receptor activation and subsequent suppression of neuronal ATP release. We investigated the effect of A1 receptor activation and downstream cAMP-dependent pathways on nerve-mediated ATP and ACh release, and detrusor contraction in mouse detrusor. Bladders from male C57BL/6 mice (12 wk) were used for in vitro experiments. Upon electrical field stimulation of intact preparations (detrusor and mucosal layers), ATP or ACh release was measured simultaneously with tension recordings. Activation of A1 receptors by adenosine or exogenous agonists reduced the lower frequency component of nerve-mediated contractions and neuronal ATP release. The A1 receptor antagonist abolished these effects. A1 receptor activation inhibits adenylyl cyclase (AC) activity and cAMP generation. The effect of A1 receptor activation was mimicked by a PKA antagonist but not by modulators of exchange proteins activated by cAMP, demonstrating that modulation of nerve-mediated ATP release is via PKA. Adenosine had no effect on ACh release or the higher frequency component of nerve-mediated contractions. Differential regulation of neurotransmitter release is possible at the detrusor nerve-muscle junction, as demonstrated by A1 receptor activation, and downstream inhibition of AC, cAMP generation, and PKA. The ability to specifically attenuate ATP release offers a potential to target purinergic motor pathways enhanced in overactive bladder pathologies.

Permixon®, hexane-extracted Serenoa repens, inhibits human prostate and bladder smooth muscle contraction and exerts growth-related functions in human prostate stromal cells

AimsRecently, the European Association of Urology recommended hexane-extracted fruit of Serenoa repens (HESr) in their guidelines on management of non-neurogenic male lower urinary tracts symptoms (LUTS). Despite previously lacking recommendations, Permixon® is the most investigated HESr in clinical trials, where it proved effective for male LUTS. In contrast, underlying mechanisms were rarely addressed and are only marginally understood. We therefore investigated effects of Permixon® on human prostate and detrusor smooth muscle contraction and on growth-related functions in prostate stromal cells. Main methodsPermixon® capsules were dissolved using n-hexane. Contractions of human prostate and detrusor tissues were induced in organ bath. Proliferation (EdU assay), growth (colony formation), apoptosis and cell death (flow cytometry), viability (CCK-8) and actin organization (phalloidin staining) were studied in cultured human prostate stromal cells (WPMY-1). Key findingsPermixon® inhibited α1-adrenergic and thromboxane-induced contractions in prostate tissues, and methacholine-and thromboxane-induced contractions in detrusor tissues. Endothelin-1-induced contractions were not inhibited. Neurogenic contractions were inhibited in both tissues in a concentration-dependent manner. In WPMY-1 cells, Permixon® caused concentration-dependent breakdown of actin polymerization, inhibited colony formation, reduced cell viability, and proliferation, without showing cytotoxic or pro-apoptotic effects. SignificanceOur results provide a novel basis that allows, for the first time, to fully explain the ubiquitous beneficial effects of HESr in clinical trials. HESr may inhibit at least neurogenic, α1-adrenergic and thromboxane-induced smooth muscle contraction in the prostate and detrusor, and in parallel, prostate stromal cell growth. Together, this may explain symptom improvements by Permixon® in previous clinical trials.

Impairment of urinary bladder mechanical properties in rat model of type 2 diabetes.

The urinary bladder is a mechanosensitive organ that accumulates, stores, and expels considerable amounts of fluid. While the neuronal bladder control via the CNS is well defined, the data on the mechanisms of local mechanical sensitivity of the bladder wall are either insufficient or contradictory. Here we compared the mechanical properties of bladder wall of normal rats and rats with modeled type 2 diabetes (T2D). T2D was modeled in 3-month-old Wistar male rats by combined administration of nicotinamide (230 mg/kg) and streptozotocin (65 mg/kg). Cystometry of isolated, denervated whole bladders and stress-strain tensiometry on detrusor smooth muscle (DSM) strips were used to assess the mechanical properties of bladder wall tissues from control and diabetic animals on 10th week after induction. The pressure-volume cystometrograms of both control and T2D bladders featured a quasi plateau between ascending sections. T2D cystometrograms revealed markedly elevated intravesicular pressure (~100% at 1 ml) and a shortened plateau, consistent with decreased bladder wall elasticity and reduced structural bladder capacity versus control. Experiments on urothelium-intact and urothelium-devoid DSM strips have shown that the decrease of bladder walls elasticity in T2D can be explained by the switch of stretched urothelium from inducing DSM relaxation to inducing DSM contraction due to a change in the prevalent release of contractile versus relaxing urothelial factor(s). The decreased elasticity of the bladder walls in T2D results from alterations in urothelium-dependent mechanosensory mechanisms. Elevated intravesical pressure in T2D may contribute to urge incontinence and/or symptoms of upper urinary tract damage.

Frequency-dependent characteristics of nerve-mediated ATP and acetylcholine release from detrusor smooth muscle.

What is the central question of this study? Is the frequency dependence of co-transmitter release from postganglionic nerve fibres different for each transmitter? What is the main finding and its importance? Release of co-transmitters from the parasympathetic supply to detrusor smooth muscle can be independently regulated. This offers a targeted drug model to reduce selectively the release of transmitter associated with human pathologies (ATP) and may also be applicable to other smooth muscle-based disorders of visceral tissues. Nerve-mediated contractions of detrusor smooth muscle are mediated by acetylcholine (ACh) and ATP release in most animals. However, with the normal human bladder, only ACh is a functional transmitter, but in benign pathologies such as overactive bladder (OAB), ATP re-emerges as a secondary transmitter. The selective regulation of ATP release offers a therapeutic approach to manage OAB, in contrast to current primary strategies that target ACh actions. However, the release characteristics of nerve-mediated ACh and ATP are poorly defined and this study aimed to measure the frequency dependence of ACh and ATP release and determine if selective regulation of ATP or ACh was possible. Experiments were carried out in vitro with mouse detrusor with nerve-mediated ATP and ACh release measured simultaneously with tension recording. ATP was released in two frequency-dependent components, both at lower frequencies (mid-range 0.4 and 5.5Hz stimulation) compared to a single compartment release of ACh at 14Hz. Intervention with the phosphodiesterase type-5 inhibitor sildenafil attenuated ATP release, equally from both components, but had no effect on ACh release. These data demonstrate that nerve-mediated ACh and ATP release characteristics are distinct and may be separately manipulated. This offers a potential targeted drug model to manage benign lower urinary tract conditions such as OAB.

Investigation of the effect of trazodone on spontaneous and acetylcholine-induced bladder detrusor smooth muscle contractions

Objective: The management of psychiatric patients is often complicated by medical comorbidities, complex pharmacological regimens, and side effects secondary to these regimens. In the current study, it was aimed to investigate the effects and mechanism of action of trazodone on rat bladder smooth muscle contractility in vitro. Method: Sixteen adult male Wistar albino rats were euthanized by the cervical dislocation method following ether anesthesia. Two muscle strips of 2 × 10 mm in size were prepared vertically from the bladder by opening a longitudinal incision from the neck of the bladder in the direction of the apex. The strips were tied properly and hung in the organ bath. All contraction amplitudes and frequencies were recorded. After a 45-min adaptation period, contractions were induced by applying 105 M acetylcholine (ACh) to all spontaneously contracting bladder strips. After 20 min, doses of trazodone (109 M vs 103 M) were given cumulatively. The resulting effects were recorded. Results: The groups were compared within themselves: a significant difference was found between the initial tensions in the group with ACh-induced contractions and the tensions after the administration of trazodone at 109, 108, 107, 106, 105, 104, and 103 M doses (p < 0.0001). In the group with spontaneous contractions, a significant difference was found between the initial tensions and the tensions after the administration of trazodone at 109, 108, 105, and 104 M doses (p < 0.0001). Conclusion: Results showed that trazodone had a significant inhibitory effect on bladder smooth muscle contractions in vitro, especially at concentrations of 104 and 103 M.

ATP as a cotransmitter in sympathetic and parasympathetic nerves - another Burnstock legacy

Geoff Burnstock created an outstanding scientific legacy that includes identification of adenosine 5′-triphosphate (ATP) as an inhibitory neurotransmitter in the gut, the discovery and characterisation of a large family of purine and uridine nucleotide-sensitive ionotropic P2X and metabotropic P2Y receptors and the demonstration that ATP is as an excitatory cotransmitter in autonomic nerves. The evidence for cotransmission includes that: 1) ATP is costored with noradrenaline in synaptic vesicles in postganglionic sympathetic nerves innervating smooth muscle tissues, including the vas deferens and most arteries. 2) When coreleased with noradrenaline, ATP acts at postjunctional P2X1 receptors to elicit depolarisation, Ca2+ influx, Ca2+ sensitisation and contraction. 3) ATP is also coreleased with acetylcholine from postganglionic parasympathetic nerves innervating the urinary bladder, where it stimulates postjunctional P2X1 receptors, and a second, as yet unidentified site to evoke contraction of detrusor smooth muscle. In both systems membrane-bound ecto-enzymes and soluble nucleotidases released from postganglionic nerves dephosphorylate ATP and so terminate its neurotransmitter actions. Currently, the most promising potential area of therapeutic application relating to cotransmission is treatment of dysfunctional urinary bladder. This family of disorders is associated with the appearance of a purinergic component of neurogenic contractions. This component is an attractive target for drug development and targeting it may be a rewarding area of research.

Purinergic signalling in the urinary bladder - When function becomes dysfunction.

Knowledge of the participation of ATP and related purines in urinary tract physiology has been established over the last five decades through the work of many independent groups, inspired by, and building on the pioneering studies of Professor Geoffrey Burnstock and his coworkers. As part of a series of reviews in this tribute edition, the present article summarises our current understanding of purines and purinergic signalling in modulating and regulating urinary tract function. Purinergic mechanisms underlying the origin of bladder pain; sensations of bladder filling and urinary tract motility; and regulation of detrusor smooth muscle contraction are described, encompassing the relevant history of discovery and consolidation of knowledge as methodologies and pharmacological tools have developed. We consider normal physiology, including development and ageing and then move to pathophysiology, discussing the causal and consequential contribution of purinergic signalling mechanism and their constituent components (receptors, signal transduction, effector molecules) to bladder dysfunction.

Inhibition of Female and Male Human Detrusor Smooth Muscle Contraction by the Rac Inhibitors EHT1864 and NSC23766

IntroductionLower urinary tract symptoms (LUTS) due to overactive bladder (OAB) are caused by spontaneous detrusor contractions. Medical treatment with muscarinic receptor antagonists or β3-adrenoceptor agonists aims to inhibit detrusor contractions, but overall results are unsatisfactory. Consequently, improved understanding of bladder smooth muscle contraction and identification of novel compounds for its inhibition are needed to develop alternative options. A role of the GTPase Rac1 for smooth muscle contraction has been reported from the prostate, but is unknown in the human detrusor. Here, we examined effects of the Rac inhibitors NSC23766, which may also antagonize muscarinic receptors, and EHT1864 on contraction of human detrusor tissues.MethodsFemale and male human detrusor tissues were obtained from radical cystectomy. Effects of NSC23766 (100 µM) and EHT1864 (100 µM) on detrusor contractions were studied in an organ bath.ResultsElectric field stimulation induced frequency-dependent contractions of detrusor tissues, which were inhibited by NSC23766 and EHT1864. Carbachol induced concentration-dependent contractions. Concentration response curves for carbachol were shifted to the right by NSC23766, reflected by increased EC50 values, but unchanged Emax values. EHT1864 reduced carbachol-induced contractions, resulting in reduced Emax values for carbachol. The thromboxane analog U46619 induced concentration-dependent contractions, which remained unchanged by NSC23766, but were reduced by EHT1864.ConclusionsNSC23766 and EHT1864 inhibit female and male human detrusor contractions. NSC23766, but not EHT1864 competitively antagonizes muscarinic receptors. In addition to neurogenic and cholinergic contractions, EHT1864 inhibits thromboxane A2-induced detrusor contractions. The latter may be promising, as the origin of spontaneous detrusor contractions in OAB is noncholinergic. In vivo, both compounds may improve OAB-related LUTS.

Signal transduction pathways of detrusor smooth muscle contraction evoked by prostanoids and isoprostanes in murine urinary bladder

The overactive bladder (OAB) is a clinical condition characterized by symptoms of frequency, urgency with/without incontinence, with a prevalence of 16% in the developed countries. The current pharmacological treatment mainly consists of anticholinergic drugs with several side effects. According to prior experimental results the arachidonic acid (AA) derivate prostanoids and isoprotanes, the latter produced non‐enzymatically during oxidative stress, are not only biomarkers of systemic oxidative stress, but are excreting in the urine and might act directly on the detrusor muscle leading to detrusor overactivity.Our aim was to examine the effects and the signal transduction pathways of prostanoids and isoprotanes in the urinary bladder smooth muscle, and potentially provide theoretical basis for the development of more specific medication of OAB with less adverse effects.Detrusor muscle strips were prepared from wild type (C57BL/6) and knockout mice, deficient for the thromboxane receptor (TP) or the α‐subunits of heterotrimeric G proteins (Gαq/11‐KO, Gα12/13‐KO) without urothelium under dissection microscope. Contraction force was measured by myograph under isometric conditions and normalized to the reference contractions evoked by 124 mM K+.The prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α), as well as the isoprostane 8‐epi‐PGE2 and 8‐iso‐PGF2α evoked contraction in the urinary bladder strips. The effect of the prostanoids was decreased, and the effect of the isoprostanes was abolished in the strips of TP KO mice, suggesting that the effect of the prostanoids is mediated partially, whereas that of the isoprostanes mainly by the TP. The TP receptor selective agonist U‐46619 evoked dose‐dependent contraction in the bladder strips, and neither the cholinerg antagonist atropine, nor the purinerg P2X‐antagonist pyridoxalphosphate‐6‐azophenyl‐2',4'‐disulfonic acid (PPADS) decreased the responses, indicating that the TP agonist has a direct effect on the detrusor muscle. The responses evoked by U‐46619 were comparable to the effect evoked by cholinergic agonist, carbachol. The contraction responses were decreased in the strips of the Gα12/13‐KO mice. Correspondingly, the responses evoked by the prostanoids and isoprostanes were reduced by the Rho‐kinase (ROCK) inhibitor Y‐27632. In the strips of the Gαq/11‐KO mice, the responses were also decreased and in the presence of Y‐27632 abolished completely.In conclusion, the examined prostanoids and isoprostanes evokes contraction acting directly on the detrusor muscle. These responses are mediated mainly by the TP receptor and are linked to the Gαq/11 and to the Gα12/13‐Rho‐ROCK intracellular signaling pathways in the murine urinary bladder. The Gα12/13‐Rho‐ROCK signaling pathway may provide a novel, more specific pharmacological target with less adverse effects in the treatment of OAB.Support or Funding InformationK‐112964, K‐125174 and NVKP‐16‐1‐2016‐0042 from the Hungarian National Research, Development and Innovation Office and EFOP‐3.6.3‐VEKOP‐16‐2017‐00009.

Effect of Entresto (valsartan+sacubitril) in the cardiac function and contractile response of detrusor and corpus cavernosum smooth muscles of heart failure rats.

IntroductionHeart failure (HF) has been closely related to the development of lower urinary tract symptoms (LUTS) and erectile dysfunction (ED). New pharmacological therapies aims to restore the neurohumoral balance, among these new proposals, stands out LCZ696 (Entresto), which consists of the association of the Valsartan (ANG‐II receptor antagonist, AT1R) with a Sacubitril (neprilysin inhibitor). However, there is no evidence evaluating the neurohumoral balance and the impact of Entresto on ED and LUTS associated with HF.ObjectiveWe aimed to Investigated the effect of in vitro treatment with Entresto (valsartan+sacubitril) on the contractile response of detrusor and corpus cavernosum (CC) muscles of control rats, as well as the effect of long‐term treatment with Entresto in the cardiovascular, urinary and erectile dysfunctions of HF rats.MethodsFor in vitro protocols, response concentration curves were constructed to phenylephrine (PHE; CC) and carbachol (CCh; detrusor), as well as, response frequency curves to electrical field stimulation (EFS; CC and detrusor) in the absence or presence of valsartan (10μM), sacubitril (10μM) or both drugs. For the chronic protocols, HF was surgically induced through the FAC model, and after 8 weeks, these animals were submitted to cardiac function evaluation (echocardiogram) and subdivided into 3 groups: Sham, HF and HF/Entresto (60mg/day for more 4 weeks). Response concentration curve were constructed to PHE (CC) and CCh (detrusor), and response frequency curve to EFS (CC and detrusor) for all groups. Culture human umbilical vein endothelial cell (HUVEC) monolayers were used to model the endothelial barrier. Electric cell‐substrate impedance sensing (ECIs) was used to study the contribution of valsartan, sacubitril or both drugs (10μM, respect.) to endothelial barrier function.ResultsIn vitro, valsartan, sacubitril or both drugs did not change the contractile responses in the CC or detrusor, independent of stimulated used in control animals. The HF animals presented both reduced of ejection and shortening fraction (p&lt;0.05), as well present cardiac hypertrophy (p&lt;0.05) when compared to the Sham group. Long‐term treatment with Entresto restored the ejection and shortening fraction in the HF/Entresto group (p&lt;0.05). In the CC of HF group the contractile response mediated by PE and EFS (p&lt;0.05) were increased compared to Sham group, being this hypercontractility restored by the Entresto treatment (p&lt;0.05). CCh and EFS induced detrusor smooth muscle contractions, which were greater in HF rats than in Sham rats. The treatment with Entresto restore the detrusor hypercontractility of HF rats (p&lt;0.05). Additionally, only the valsartan enhance the barrier function (P&lt;0.05) of HUVEC monolayer.ConclusionOur data provide evidence that Entresto chronic treatment, restore the detrusor‐ and CC‐contractile response due the improved of endothelial barrier and cardiovascular function in rats with HF, suggesting new multi‐therapeutic properties.Support or Funding InformationFAPESP 2011/21095‐4, CNPq 423987/2016‐0 and NIH R01GM20774

Characterisation of nerve-mediated ATP release from bladder detrusor muscle and its pathological implications.

This study aims to characterise the molecular mechanisms that determine variability of atropine resistance of nerve-mediated contractions in human and guinea pig detrusor smooth muscle. Atropine resistance of nerve-mediated contractions and the role of P2X1 receptors, were assessed in isolated preparations from guinea pigs and also humans with or without overactive bladder syndrome, from which the mucosa was removed. Nerve-mediated ATP release was measured directly with amperometric ATP-sensitive electrodes. Ecto-ATPase activity of guinea pig and human detrusor samples was measured in vitro by measuring the concentration-dependent rate of ATP breakdown. The transcription of ecto-ATPase subtypes in human samples was measured by qPCR. Atropine resistance was greatest in guinea pig detrusor, absent in human tissue from normally functioning bladders, and intermediate in human overactive bladder. Greater atropine resistance correlated with reduction of contractions by the ATP-diphosphohydrolase apyrase, directly implicating ATP in their generation. E-NTPDase-1 was the most abundantly transcribed ecto-ATPase of those tested, and transcription was reduced in tissue from human overactive, compared to normal, bladders. E-NTPDase-1 enzymic activity was inversely related to the magnitude of atropine resistance. Nerve-mediated ATP release was continually measured and varied with stimulation frequency over the range of 1-16Hz. Atropine resistance in nerve-mediated detrusor contractions is due to ATP release and its magnitude is inversely related to E-NTPDase-1 activity. ATP is released under different stimulation conditions compared with ACh, implying different routes for their release.

Pharmacological Characteristics of Anxiolytics on Acetylcholine-Induced Contractions in Rat Detrusor Smooth Muscle

Introduction: Benzodiazepine anxiolytics are believed to cause urination disorders due to their anticholinergic effects. Objective: This study was carried out to investigate the potential inhibitory effects of 15 clinically available anxiolytics in Japan on acetylcholine (ACh)-induced contractions in rat detrusor smooth muscle (DSM) to predict whether these anxiolytics could induce urination disorders. Methods: ­Effects of anxiolytics on contractions induced by ACh and 80 mmol/L KCl solution in rat DSM and effects of anxiolytics on specific binding of [N-methyl-³H]scopolamine ([³H]NMS) in mouse cerebral cortex were investigated. Results and Conclusions: ACh-induced contractions in rat DSM were inhibited by clotiazepam and diazepam (benzodiazepine anxiolytics) at concentrations that were clinically relevant. These contractions were also significantly inhibited by paroxetine, escitalopram (selective serotonin reuptake inhibitors ­[SSRIs]), and hydroxyzine (a histamine H₁ receptor antagonist), albeit at concentrations that substantially exceeded clinically achievable blood levels. At a concentration of 10^–5 mol/L, paroxetine, escitalopram, and hydroxyzine inhibited 80 mmol/L high-KCl solution-induced rat DSM contractions but not clotiazepam and diazepam. Paroxetine, escitalopram, and hydroxyzine also inhibited specific binding of [³H]NMS in mouse cerebral cortex but clotiazepam and diazepam did not. In contrast to the effects of the abovementioned anxiolytics, ACh-induced contractions were not significantly affected by tofisopam, alprazolam, lorazepam, bromazepam, oxazolam, chlordiazepoxide, clonazepam, ethyl loflazepate (benzodiazepine anxiolytics), fluvoxamine (an SSRI), or tandospirone (a serotonin 5-HT_1A receptor agonist). These findings suggest that most clinically used anxiolytics are not likely to result in anticholinergic-induced urination disorders within their clinically achievable blood concentration ranges. However, clotiazepam and diazepam may induce urination disorders within their clinical dose ranges via nonanticholinergic inhibition of DSM contractility.

Bladder Dysfunction in an Obese Zucker Rat: The Role of TRPA1 Channels, Oxidative Stress, and Hydrogen Sulfide.

Purpose This study investigates whether functionality and/or expression changes of transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels, oxidative stress, and hydrogen sulfide (H2S) are involved in the bladder dysfunction from an insulin-resistant obese Zucker rat (OZR). Materials and Methods Detrusor smooth muscle (DSM) samples from the OZR and their respective controls, a lean Zucker rat (LZR), were processed for immunohistochemistry for studying the expression of TRPA1 and TRPV1 and the H2S synthase cystathionine beta-synthase (CBS) and cysthathionine-γ-lyase (CSE). Isometric force recordings to assess the effects of TRPA1 agonists and antagonists on DSM contractility and measurement of oxidative stress and H2S production were also performed. Results Neuronal TRPA1 expression was increased in the OZR bladder. Electrical field stimulation- (EFS-) elicited contraction was reduced in the OZR bladder. In both LZR and OZR, TRPA1 activation failed to modify DSM basal tension but enhanced EFS contraction; this response is inhibited by the TRPA1 blockade. In the OZR bladder, reactive oxygen species, malondialdehyde, and protein carbonyl contents were increased and antioxidant enzyme activities (superoxide dismutase, catalase, GR, and GPx) were diminished. CSE expression and CSE-generated H2S production were also reduced in the OZR. Both TRPV1 and CBS expressions were not changed in the OZR. Conclusions These results suggest that an increased expression and functionality of TRPA1, an augmented oxidative stress, and a downregulation of the CSE/H2S pathway are involved in the impairment of nerve-evoked DSM contraction from the OZR.

Urinary dysfunction in transgenic sickle cell mice: model of idiopathic overactive bladder syndrome.

Voiding abnormalities are common among the sickle cell disease (SCD) population, among which overactive bladder (OAB) syndrome is observed at rates as high as 39%. Although detrusor overactivity is the most common cause of OAB, its molecular pathophysiology is not well elucidated. The nitric oxide (NO) signaling pathway has been implicated in the regulation of lower genitourinary tract function. In the present study, we evaluated the role of the NO signaling pathway in voiding function of transgenic SCD mice compared with combined endothelial and neuronal NO synthase gene-deficient mice, both serving as models of NO deficiency. Mice underwent void spot assay and cystometry, and bladder and urethral specimens were studied using in vitro tissue myography. Both mouse models exhibited increased void volumes; increased nonvoiding and voiding contraction frequencies; decreased bladder compliance; increased detrusor smooth muscle contraction responses to electrical field stimulation, KCl, and carbachol; and increased urethral smooth muscle relaxation responses to sodium nitroprusside compared with WT mice. In conclusion, our comprehensive behavioral and functional study of the SCD mouse lower genitourinary tract, in correlation with that of the NO-deficient mouse, reveals NO effector actions in voiding function and suggests that NO signaling derangements are associated with an OAB phenotype. These findings may allow further study of molecular targets for the characterization and evaluation of OAB.

Mirabegron induces relaxant effects via cAMP signaling-dependent and -independent pathways in detrusor smooth muscle.

We previously found that mirabegron exerts a relaxant effect in the presence of the β3 -adrenoceptor antagonist SR58894A during carbachol-induced contraction in human and pig detrusor. The aim of this study was to explore the possible mechanism underlying the relaxant effects of mirabegron using detrusor smooth muscle. Human tissue was obtained from urinary bladders of patients undergoing radical cystectomy at Kyushu University and Harasanshin Hospital. Pig tissue was obtained from an abattoir. Tension force (organ bath experiments) was measured in intact or permeabilised (α-toxin or β-escin) detrusor smooth muscle strips. The contribution of cAMP-dependent signaling and the inhibition of Ca2+ sensitization to the relaxant effects of mirabegron were characterized using 1 μM SR58894A, 100 μM SQ22536 (an adenylyl cyclase inhibitor), 10 μM H-89 (a protein kinase [PK] A inhibitor), 10 μM Y-27632 (a selective Rho kinase inhibitor), and 10 μM GF-109203X (a selective PKC inhibitor). 30 μM Mirabegron impaired carbachol (0.03-1 μM)-induced contraction in human detrusor smooth muscle. SR58894A only partially attenuated the relaxant effects of mirabegron in human and pig detrusor strips precontracted with 1 μM carbachol. In α-toxin-permeabilized detrusor strips, tension force at 1 μM [Ca2+ ]i was decreased by mirabegron in a concentration-dependent manner. The relaxant effect of mirabegron was only slightly attenuated by H-89 and not significantly affected by SQ22536. Y-27632 potentiated the relaxation response to mirabegron, but attenuated responses to cAMP; GF-109203X had little effect. Mirabegron but not cAMP had a notable relaxant effect in the pig detrusor smooth muscle permeabilized with β-escin. Mirabegron-induced relaxation of pig and human detrusor smooth muscle occurs via both a β3 -adrenoceptor/cAMP-dependent and -independent pathway.