Detrusor Muscle Strips Research Articles

Age-dependent effects of the β3 adrenoceptor agonist CL316,243 on human and rat detrusor muscle strips

Motility of detrusor smooth muscle includes adrenergic relaxation and cholinergic contraction. Since the latter may be deregulated in overactive bladder (OAB) pathophysiology, anticholinergics are the standard therapy but occasionally less tolerated due to side effects such as dry mouth and constipation. β3 adrenoceptor agonists also alleviate OAB symptoms by relaxing the detrusor muscle. Their age dependence, however, is far from understood. To address this issue, we induced contractions with KCl (60 mM) and carbachol (from 10 nM to 100 μM) in the presence of the β3 adrenoceptor agonist CL316,243 (from 0.1 to 10 μM) in both human and rat muscle strips. Our results confirmed that both contractions were attenuated by β3 adrenoceptor activation in both species, but with differing age dependence. In humans, specimens from mid-life subjects showed a significantly more pronounced effect of CL316,243 in attenuating carbachol-induced contractions than those from aged subjects (Cohen’s d of maximal attenuation: 1.82 in mid-life versus 0.13 in aged) without altering EC50. Conversely, attenuation of KCl responses by CL316,243 increased during ageing (Spearman correlation coefficient = -0.584, P<0.01). In rats, both KCl- and carbachol-induced contractions were significantly more attenuated by CL316,243 in samples from adolescent as compared to aged samples. Immunohistochemistry in human detrusor sections proved β3 adrenoreceptor abundance to remain unaltered during ageing. In conclusion, our findings suggest differential age-dependent changes in human β3 adrenoceptor-dependent attenuation of detrusor contraction in terms of electromechanical versus pharmacomechanical coupling; they may help understand the differential responsiveness of OAB patients to β3 agents.

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 &amp;lt; 0.0001). The contractile responses induced by EFS were significantly inhibited by preincubation with PF-573228 at 8, 16, and 32 Hz (P &amp;lt; 0.05) or latrunculin B at 16 and 32 Hz (P &amp;lt; 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.

Focal adhesion kinase activation is involved in contractile stimulation-induced detrusor muscle contraction in mice

Recent studies suggested smooth muscle contraction may involve mechanisms besides the myosin regulatory light chain (MLC) phosphorylation-induced actomyosin crossbridge cycling. This study aims to determine if focal adhesion kinase (FAK) activation is involved in mouse detrusor muscle contraction. The mouse detrusor muscle strips were preincubated with PF-573228 (2 μM), latrunculin B (1 μM), or the same volume of vehicle (DMSO) for 30 min. 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 phosphorylated FAK (p-FAK) and MLC (p-MLC) levels were measured in the detrusor strips stimulated with CCh (10 μM) after incubation with PF-573228 or vehicle (DMSO) compared to those with vehicle incubation but without CCh stimulation. KCl-induced contractile responses decreased significantly after incubation with PF-573228 or latrunculin B compared to the corresponding vehicle-treated strips (p < 0.0001). The contractile responses induced by EFS were markedly 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 lower than the corresponding vehicle group (p = 0.0021 and 0.0003, respectively). Western blot examination showed that CCh stimulation enhanced the expression of p-FAK and p-MLC, while preincubation with PF-573228 prevented the increase of p-FAK but not p-MLC. In conclusion, FAK activation involves tension development induced by contractile stimulation in the mouse detrusor muscle. This effect is likely caused by promoting actin polymerization rather than elevating MLC phosphorylation.

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+.

TrkA inhibition alleviates bladder overactivity in cyclophosphamide-induced cystitis by targeting hyperpolarization-activated cyclic nucleotide-gated channels.

To investigate the potential of Tropomyosin receptor kinase A (TrkA) for the treatment of interstitial cystitis/ bladder pain syndrome (IC/BPS). Sixty-four female rats were randomly assigned to the control and cyclophosphamide (CYP) groups. Quantitative reverse transcription polymerase chain reaction was utilized to detect the mRNA level of TrkA. Western blot analysis was used to measure the protein levels of TNF-α, IL-6, and TrkA. Immunostaining was used to detect the expression of TrkA in bladder sections. Contractility studies and urodynamic measurements were utilized to test the spontaneous contractions of detrusor muscle strips and the global bladder activity, respectively. Rat models of chronic cystitis were successfully established. The mRNA and protein levels of TrkA were significantly increased in the bladders of CYP-treated rats. Also, results of immunohistochemical staining and immunofluorescence staining showed that increased TrkA expression in the CYP group was mainly observed in the urothelium layer and bladder interstitial Cajal-like cells (ICC-LCs) but not in the detrusor smooth muscle cells. The specific inhibitor of TrkA, GW441756 (10 μM), significantly suppressed the robust spontaneous contractions of detrusor muscle strips in the CYP group and alleviated the overall bladder overactivity of CYP-treated rats. However, the inhibitory effects of GW441756 (10 μM) on the spontaneous contractions of detrusor muscle strips and the overall bladder activity were eliminated after pretreatments with the specific blocker of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, ZD7288 (50 μM). Our results suggested that increased TrkA expression during chronic cystitis promotes the development of bladder overactivity by targeting the HCN channels.

Vinpocetine Ameliorates Metabolic-Syndrome-Associated Bladder Overactivity in Fructose-Fed Rats by Restoring Succinate-Modulated cAMP Levels and Exerting Anti-Inflammatory Effects in the Bladder Detrusor Muscle

Succinate and its receptor, the G protein-coupled receptor 91 (GPR91), have pathological implications in metabolic syndrome (MetS) and its associated bladder dysfunction, particularly in decreasing bladder cAMP levels and promoting proinflammation. Using fructose-fed rats (FFRs), a rat model of MetS, we investigate the effects of vinpocetine (a phosphodiesterase-1 inhibitor) and celecoxib (a selective cyclooxygenase-2 inhibitor) on MetS-associated bladder overactivity. Phenotypes of the overactive bladder, including increased micturition frequency and a shortened intercontractile interval in cystometry, were observed in FFRs, together with elevated succinate levels in the liver and serum and the downregulation of GPR91 in the liver and urinary bladder. Treatments with vinpocetine and celecoxib improved tissue fibrosis and ameliorated the overexpression of the inflammatory cytokines, such as IL-1β, in the liver and bladder. In bladder organ bath studies, vinpocetine, but not celecoxib, treatment restored the contraction and relaxation responses of the detrusor muscle strip in response to KCl, carbachol, and forskolin stimulation. At a molecular level, vinpocetine and celecoxib treatments modulated the downstream messengers of GPR91 (i.e., ERK1/2 and JNK), suppressed NF-κB and IL-1β expressions in the bladder, and prevented the fibrogenesis observed in FFRs. The exogenous application of succinate to a bladder organ bath significantly reduced the forskolin-induced cAMP production by the detrusor muscle, which was notably restored in the presence of vinpocetine. Together, these results suggest that vinpocetine may alleviate the MetS-associated bladder overactivity by restoring the succinate-modulated detrusor cAMP production and exerting the anti-inflammatory effects in the bladder detrusor muscle.

Dog and human bladders have different neurogenic and nicotinic responses in inner versus outer detrusor muscle layers.

The aim of this study was to investigate layer and species variations in detrusor muscle strip responses to myogenic, neurogenic, and nicotinic, and muscarinic receptor stimulations. Strips from bladders of 9 dogs and 6 human organ transplant donors were dissected from inner and outer longitudinal muscle layers, at least 1 cm above urethral orifices. Strips were mounted in muscle baths and maximal responses to neurogenic stimulation using electrical field stimulation (EFS) and myogenic stimulation using potassium chloride (KCl, 120 mM) determined. After washing and re-equilibration was completed, responses to nicotinic receptor agonist epibatidine (10 μM) were determined followed by responses to EFS and muscarinic receptor agonist bethanechol (30 μM) in continued presence of epibatidine. Thereafter, strips and full-thickness bladder sections from four additional dogs and three human donors were examined for axonal density and intramural ganglia. In dog bladders, contractions to KCl, epibatidine, and bethanechol were 1.5- to 2-fold higher in the inner longitudinal muscle layer, whereas contractions to EFS were 1.5-fold higher in the outer (both pre- and post-epibatidine). Human bladders showed 1.2-fold greater contractions to epibatidine in the inner layer and to EFS in the outer, yet no layer differences to KCl or bethanechol were noted. In both species, axonal density was 2- to 2.5-fold greater in the outer layer. Dogs had more intramural ganglia in the adventitia/serosa layer, compared with more internal layers and to humans. These findings indicate several layer-dependent differences in receptor expression or distribution, and neurogenic responses in dog and human detrusor muscles, and myogenic/muscarinic differences between dog versus humans.

The Effects of 1,1-Dimethylbiguanide Hydrochloride (Metformin) on Detrusor Muscle Contractile Response in Ovariectomized Female Rats

Aim: Menopause is defined as the depletion of the ovarian follicular reserve followed by the cessation of menstrual cycles. It has been&#x0D; reported that gonadal steroid hormones play an important role in bladder function in women. Changes in urine pattern including&#x0D; overactive bladder, stress incontinence and recurrent urinary tract infections occur as a result of menopause. 1,1-dimethylbiguanide&#x0D; hydrochloride, metformin, (MET) is an oral anti-diabetic drug used to reduce hepatic glucose production and peripheral insulin&#x0D; resistance. Recent studies have revealed that MET has a protective effects in diabetes induced bladder dysfunction. The aim of this&#x0D; study was to test the therapeutic potential of MET in detrusor contractile function of ovariectomized (OVX) female rats.&#x0D; Material and Methods: Bilateral ovariectomy was performed to eliminate endogenous gonadal steroids secretion. Four groups are&#x0D; designed with 8 animals in each group: Control, MET-administered control, OVX, and MET-administered OVX groups. MET (25 mg/&#x0D; kg) was administered daily by oral gavage for 14 days. Contractile activity of isolated bladder muscle strips were evaluated in vitro organ&#x0D; bath. The contractile responses of detrusor strips were determined using different doses of carbachol (10-8-10-2M) and purinergic&#x0D; agonist ATP. The relaxation response of strips were determined by isoproterenol&#x0D; Results: The contractile responses of detrusor muscle strips to carbachol at doses 10-5-10-2 M were decreased in the OVX group&#x0D; compared to control and MET treated control groups. MET treatment partially reversed the reduction in OVX-induced contractile&#x0D; responses at 10-2 and 10 -3 M carbachol doses. There were no statistically significant difference in relaxation response between the&#x0D; experimental groups.&#x0D; Conclusion: Our findings suggest that treatment with MET could be the new potential therapeutic agent against bladder dyfunction&#x0D; in postmenopausal women. Further studies are needed for the therapeutic potential of MET in detrusor dysfunction induced by&#x0D; menopause.

Human Urine-Derived Stem Cells Improve Partial Bladder Outlet Obstruction in Rats: Preliminary Data and microRNA-mRNA Expression Profile

Partial bladder outlet obstruction (pBOO) often results in bladder tissue inflammation and remodeling. As human urine-derived stem cells (USCs) have demonstrated therapeutic benefits, we used a rat model to investigate the effect of USCs on bladder function and explore the miRNA and gene expression profiles in bladder tissue using RNA sequencing. Eighteen rats were assigned to a sham surgery group, pBOO group, and pBOO+USC group (six biweekly treatments). Routine urodynamic monitoring, analysis of detrusor muscle strips, and pathophysiology assessments were conducted. Finally, altered miRNA and mRNA expression profiles of bladder tissue were examined using RNA sequencing and bioinformatics analysis. After USC treatment, elevated bladder compliance and maximal voiding pressure, declined end filling pressure and voided volume, and improved detrusor muscle contractility and carbachol sensitivity were found. Histology and TUNEL assay revealed reduced collagen deposition and muscle cell apoptosis in bladder tissue. The differential expression of eight miRNAs was reversed by USC treatment. Two large nodes (miR-142 and miR-9a) were identified in the miRNA-gene interaction network in the USC-treated group. The Kyoto Encyclopedia of Genes and Genomes analysis revealed enrichment of multiple significant pathways, including those involved in necroptosis and cytokine-cytokine receptor interactions. This is the first study to demonstrate the protective effect of USCs on bladder function and remodeling in pBOO rats. The miRNA and mRNA expression levels differed in the bladder of pBOO rats with and without USC treatment. Although the mechanism underlying these effects has not been fully elucidated, necroptosis and cytokine-cytokine receptor interaction-related pathways may be involved.Graphical abstract

Uroprotective effect of pantoprazole against cyclophosphamide-induced cystitis in mice.

The aim of the present study was to evaluate the potential uroprotective effect of pantoprazole (PPZ) in a mouse model of cyclophosphamide (CP)-induced hemorrhagic cystitis (HC) due to its antioxidant and anti-inflammatory properties. Balb/c mice received a single intraperitoneal (i.p.) injection of CP (300mg/kg) to induce HC. PPZ (20, 50, and 100mg/kg/day;i.p.) was administered for 3 consecutive days before the induction of HC. Mesna (30mg/kg;i.p.) was administered 20min before, 4 and 8h after CP injection to compare the protective effects of PPZ. After 24h of HC induction, the bladders were removed for functional studies, biochemical analyses, and histopathological examination. In vitro contractility studies demonstrated that CP-induced HC decreased the responsiveness of detrusor muscle strips to acetylcholine (ACh), which was reversed by PPZ pretreatment at all doses tested. However, mesna treatment was not able to improve responsiveness to ACh. Biochemical analyses showed that CP caused significant elevation of malondialdehyde (MDA), reduction of total glutathione (GSH), and increment of proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) level, which were measured in bladder homogenates. PPZ pretreatment at three doses found to be effective in reducing the CP-induced elevation of MDA and TNF-α levels. The highest dose of PPZ (100mg/kg) caused a significant increase in GSH level. CP induced severe HC with marked bladder edema and histological disturbances which were partially abolished by PPZ pretreatment. Our results indicate that PPZ pretreatment could attenuate CP-induced HC by interfering with oxidative stress and modulating proinflammatory cytokines.

Activation of β3‐adrenoceptors inhibits purinergic‐mediated contractions of the murine detrusor

β3‐adrenergic receptor (β3‐AR) agonists are used for treatment of overactive bladder, however the mechanisms underlying their effect are unclear. β3‐AR agonists inhibit cholinergic‐mediated responses of the detrusor, however the concentration required to exert this effect exceed the plasma levels achieved through therapeutic dosing of these drugs1. ATP is regarded as an excitatory neurotransmitter in the detrusor, and upregulation of purinergic signaling is considered as a causative factor in the pathogenesis of overactive bladder2. The purpose of the present study was 1) to examine the effect of β3‐AR activation on purinergic‐evoked contractions of the murine detrusor &amp; on P2XR1 currents recorded from isolated murine detrusor myocytes and 2) to investigate if purinergic responses in the detrusor were modulated by agents that activate exchange factor directly activated by cAMP (EPAC), a novel cAMP effector.Mice were killed by cervical dislocation and their bladder removed, in accordance with the DKIT Animal Care &amp; Use Committee and EU legislation (EU Directive 2010/63/EU). Using fine dissection, the urothelium was removed leaving the remaining detrusor layer. Isometric tension recordings were made from strips of detrusor muscle. Detrusor smooth muscle cells (DSMCs) were isolated by enzymatic digestion (Collagenase Type II, 5 minutes at 37°C) of detrusor tissue cut into fine pieces. The perforated patch clamp configuration was used to investigate inward currents in isolated DSMCs. Perforation was achieved using the antibiotic amphotericin B (600 μg/ml).In the presence of the muscarinic antagonist atropine (1 μM), electric field stimulation (EFS; 4 Hz) induced robust purinergic‐mediated contractions of murine detrusor strips. Therapeutic‐relevant concentrations (100 nM) of the β3‐AR agonists BRL37344 &amp; CL316,243, reduced the amplitude of EFS‐evoked detrusor contractions by 59% (n=8, p&lt;0.01) and 50% (n=6, p&lt;0.05), respectively. These effects were partially reversed by the β3‐AR antagonist, L748,337 (1 μM). The adenylyl cyclase stimulant forskolin and the PDE4 inhibitor rolipram, similarly inhibited EFS‐evoked purinergic contractions. Under voltage clamp conditions (‐60 mV), ATP (1 μM) evoked inward currents in isolated DSMCs. These currents were reduced from −1803 ± 418 pA to −514 ± 168 pA (n=6, p&lt;0.05) by BRL37344 (100 nM). Quantitative PCR experiments revealed that EPAC1 &amp; 2 subtypes were expressed in murine detrusor, with EPAC1 ~10‐fold more abundant. Application of the EPAC activator, 007‐AM, reduced the amplitude of purinergic contractions by ~48%, (n=6, p&lt;0.01), and this effect was partially reversed in the presence of the EPAC1 blocker, CE3F4.These data show that β3‐AR agonists inhibit purinergic‐mediated responses of the murine detrusor by inhibiting P2XR1 currents in detrusor myocytes, possibly by a mechanism involving activation of EPAC1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MP38-09 THE LACK OF MECHANOSENSITIVE K2P CHANNEL IS ASSOCIATED WITH MIXED VOIDING PHENOTYPE IN MICE

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology II1 Apr 2018MP38-09 THE LACK OF MECHANOSENSITIVE K2P CHANNEL IS ASSOCIATED WITH MIXED VOIDING PHENOTYPE IN MICE Ricardo Pineda, Joseph Hypolite, Sanghee Lee, Alonso Carrasco, Nao Iguchi, Randall B Meacham, and Anna P Malykhina Ricardo PinedaRicardo Pineda More articles by this author , Joseph HypoliteJoseph Hypolite More articles by this author , Sanghee LeeSanghee Lee More articles by this author , Alonso CarrascoAlonso Carrasco More articles by this author , Nao IguchiNao Iguchi More articles by this author , Randall B MeachamRandall B Meacham More articles by this author , and Anna P MalykhinaAnna P Malykhina More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1236AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder capacity is defined by the physiological response of the bladder wall to stretch. We previously reported that compliance of the bladder wall during storage phase of the micturition cycle in the human urinary bladder is regulated by a mechanosensitive TREK-1 channel, a member of the K2P potassium channel family. The use of isolated human tissue limited evaluation of in vivo role of TREK-1 on voiding function as these channels are also expressed in the nervous system and may modulate micturition via neural pathways. Therefore, in this study, we aimed to assess the role of TREK-1 channels in bladder contractility and voiding using TREK-1 knockout (KO) mice. We hypothesized that absence of TREK-1 expression could increase the number of spontaneous contractions in the urinary bladder, and alter its reposne to physiological stretch. We aimed to: 1) study the patterns of TREK-1 expression in visceral organs (kidney, urinary bladder, and colon) in WT and TREK-1 KO mice, and 2) assess the role of TREK-1 channels in bladder contractility and voiding cycle. METHODS Adult C57BL/6J wild-type (WT, N=32) and TREK-1 KO (KO, N=33) mice were used in this study. We performed immunocytochemistry labeling, endpoint RT-PCR, detrusor muscle strip contractility studies, and awake cystometry to evaluate blader function. RESULTS No changes in visceral organ morphology or tissue histology were observed between WT and TREK-1 KO animals. Isometric contractility recordings from TREK-1 KO isolated detrusor strips showed elevated basal muscle tone and enhanced spontaneous activity in response to stretch (109% vs. 61% in WT group, p=0.05). Detrusor strips from TREK-1 KO mice also generated more contractile force in response to electric field and high potassium stimulation. Interestingly, cystometric recordings from TREK-1 KO mice revealed a significant increase in the duration of inter-micturition interval, also expressed enhanced bladder capacity and increased number of non-voiding contractions in comparison to WT mice. CONCLUSIONS Our data suggest that the knockout of TREK-1 channels has dual effects on detrusor contractility and micturition patterns, most likely due to expression of TREK-1 channels on both smooth muscle and neuronal cells. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e503-e504 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Ricardo Pineda More articles by this author Joseph Hypolite More articles by this author Sanghee Lee More articles by this author Alonso Carrasco More articles by this author Nao Iguchi More articles by this author Randall B Meacham More articles by this author Anna P Malykhina More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Modeling the influence of acute changes in bladder elasticity on pressure and wall tension during filling

Tension-sensitive nerves in the bladder wall are responsible for providing bladder sensation. Bladder wall tension, and therefore nerve output, is a function of bladder pressure, volume, geometry and material properties. The elastic modulus of the bladder is acutely adjustable, and this material property is responsible for adjustable preload tension exhibited in human and rabbit detrusor muscle strips and dynamic elasticity revealed during comparative-fill urodynamics in humans. A finite deformation model of the bladder was previously used to predict filling pressure and wall tension using uniaxial tension test data and the results showed that wall tension can increase significantly during filling with relatively little pressure change. In the present study, published uniaxial rabbit detrusor data were used to quantify regulated changes in the elastic modulus, and the finite deformation model was expanded to illustrate the potential effects of elasticity changes on pressure and wall tension during filling. The model demonstrates a shift between relatively flat pressure-volume filling curves, which is consistent with a recent human urodynamics study, and also predicts that dynamic elasticity would produce significant changes in wall tension during filling. The model results support the conclusion that acute regulation of bladder elasticity could contribute to significant changes in wall tension for a given volume that could lead to urgency, and that a single urodynamic fill may be insufficient to characterize bladder biomechanics. The model illustrates the potential value of quantifying wall tension in addition to pressure during urodynamics.

Netupitant, a Potent and Highly Selective NK1 Receptor Antagonist, Alleviates Acetic Acid-Induced Bladder Overactivity in Anesthetized Guinea-Pigs.

Introduction. Tachykinins potently contract the isolated urinary bladder from a number of animal species and play an important role in the regulation of the micturition reflex. On the guinea-pig isolated urinary bladder we examined the effects of a new potent and selective NK1 receptor antagonist (netupitant) on the contractions induced by a selective NK1 receptor agonist, SP-methylester (SP-OMe). Moreover, the effects of netupitant and another selective NK1 antagonist (L-733,060) were studied in anesthetized guinea-pigs using two experimental models, the isovolumetric bladder contractions and a model of bladder overactivity induced by intravesical administration of acetic acid (AA).Methods and Results. Detrusor muscle strips were mounted in 5 mL organ baths and isometric contractions to cumulative concentrations of SP-OME were recorded before and after incubation with increasing concentrations of netupitant. In anesthetized female guinea-pigs, reflex bladder activity was examined under isovolumetric conditions with the bladder distended with saline or during cystometry using intravesical infusion of AA. After a 30 min stabilization period, netupitant (0.1–3 mg/kg, i.v.) or L-733,060 (3–10 mg/kg, i.v.) were administered. In the detrusor muscle, netupitant produced a concentration-dependent inhibition (mean pKB = 9.24) of the responses to SP-OMe. Under isovolumetric conditions, netupitant or L-733,060 reduced bladder contraction frequency in a dose-dependent manner, but neither drug changed bladder contraction amplitude. In the AA model, netupitant dose-dependently increased intercontraction interval (ICI) but had no effect on the amplitude of micturition (AM). L-733,060 dose-dependently increased ICI also but this effect was paralleled by a significant reduction of AM.Conclusion. Netupitant decreases the frequency of reflex bladder contractions without altering their amplitude, suggesting that this drug targets the afferent limb of the micturition reflex circuit and therefore may be useful clinically in treating bladder overactivity symptoms.

A pilot study to measure dynamic elasticity of the bladder during urodynamics.

Previous studies using isolated strips of human detrusor muscle identified adjustable preload tension, a novel mechanism that acutely regulates detrusor wall tension. The purpose of this investigation was to develop a method to identify a correlate measure of adjustable preload tension during urodynamics. Patients reporting urgency most or all of the time based on ICIq-OAB survey scores were prospectively enrolled in an extended repeat fill-and-empty urodynamics study designed to identify a correlate of adjustable preload tension which we now call "dynamic elasticity." Cystometric capacity was determined during initial fill. Repeat fills to defined percentages of capacity with passive emptying (via syringe aspiration) were performed to strain soften the bladder. A complete fill with active voiding was included to determine whether human bladder exhibits reversible strain softening. Five patients completed the extended urodynamics study. Intravesical pressure (pves ) decreased with subsequent fills and was significantly lower during Fill 3 compared to Fill 1 (P = 0.008), demonstrating strain softening. Active voiding after Fill 3 caused strain softening reversal, with pves in Fill 4 returning to the baseline measured during Fill 1 (P = 0.29). Dynamic elasticity, the urodynamic correlate of adjustable preload tension, was calculated as the amount of strain softening (or its reversal) per %capacity (Δaverage pves between fills/Δ%capacity). Dynamic elasticity was lost via repeat passive filling and emptying (strain softening) and regained after active voiding regulated the process (strain softening reversal). Improved understanding of dynamic elasticity in the human bladder could lead to both improved sub-typing and novel treatments of overactive bladder. Neurourol. Urodynam. 36:1086-1090, 2017. © 2016 Wiley Periodicals, Inc.

MP30-02 EFFECTS OF OMEGA-3 FATTY ACIDS IN A RAT MODEL OF ATHEROSCLEROSIS-INDUCED CHRONIC BLADDER ISCHEMIA

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II1 Apr 2016MP30-02 EFFECTS OF OMEGA-3 FATTY ACIDS IN A RAT MODEL OF ATHEROSCLEROSIS-INDUCED CHRONIC BLADDER ISCHEMIA Ji Yun Chae, Hyoung Kook Jeong, Jong Jin Park, Jong Wook Kim, Mi Mi Oh, Jae Hyun Bae, Seok Ho Kang, Hong Seok Park, Du Geon Moon, Jun Cheon, Je Jong Kim, and Jeong Gu Lee Ji Yun ChaeJi Yun Chae More articles by this author , Hyoung Kook JeongHyoung Kook Jeong More articles by this author , Jong Jin ParkJong Jin Park More articles by this author , Jong Wook KimJong Wook Kim More articles by this author , Mi Mi OhMi Mi Oh More articles by this author , Jae Hyun BaeJae Hyun Bae More articles by this author , Seok Ho KangSeok Ho Kang More articles by this author , Hong Seok ParkHong Seok Park More articles by this author , Du Geon MoonDu Geon Moon More articles by this author , Jun CheonJun Cheon More articles by this author , Je Jong KimJe Jong Kim More articles by this author , and Jeong Gu LeeJeong Gu Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1234AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Aging-related bladder dysfunction and associated lower urinary tract symptoms (LUTS) have been well documented and seem to be multifactorial. One of the factors is chronic ischemia of the bladder detrusor, resulting from atherosclerotic changes in blood vessels. The aim of this study was to evaluate the potential protective effects of omega-3 fatty acids in a rat model of atherosclerosis- induced CBI. METHODS Total 18 of 16-week old male rats were divided into three groups. The CBI group underwent endothelial injury of the iliac arteries and received 2% cholesterol diet. The omega-3 fatty acids group underwent endothelial injury also and received 2% cholesterol diet, with 2 weeks of DHA + EPA (200mg/kg/day) administration. The control group underwent sham operation and received a regular diet. After 8 weeks, in vivo cystometry and in vitro detrusor muscle strip study were performed. The plasma and bladder tissue were processed for immunohistochemical staining of oxidative stress markers. Histological examination of the bladder and iliac arteries was also done. RESULTS In cystometry, omega-3 fatty acids group showed decreased micturition frequency with prolonged intercontraction interval, increased bladder capacity compared to untreated CBI group. In the muscle strip study, omega-3 fatty acids group showed increased contractile responses compared to untreated CBI group. Plasma and bladder tissue of CBI group showed higher malondialdehyde (MDA) than in other groups. In histological examination, omega-3 fatty acids group showed lesser submucosal fibrosis of the bladder wall and also lesser degenerative change of blood vessel wall than in CBI group. CONCLUSIONS Atherosclerosis-induced CBI is associated with detrusor overactivity in cystometric study, impaired smooth muscle contractility in muscle strip study, and oxidative stress in MDA assay. Our study suggests that omega-3 fatty acids supplementation provides bladder protection against CBI on rat urinary bladder and reduces the MDA accumulation in bladder tissue caused by chronic ischemia and reperfusion. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e411-e412 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Ji Yun Chae More articles by this author Hyoung Kook Jeong More articles by this author Jong Jin Park More articles by this author Jong Wook Kim More articles by this author Mi Mi Oh More articles by this author Jae Hyun Bae More articles by this author Seok Ho Kang More articles by this author Hong Seok Park More articles by this author Du Geon Moon More articles by this author Jun Cheon More articles by this author Je Jong Kim More articles by this author Jeong Gu Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The Effect of Β3-Adrenoceptor Agonist (Brl37344) on Carbachol and Efs–Evoked Contractions of Isolated Ovine Detrusor Muscle Strip

The Effect of Β3-Adrenoceptor Agonist (Brl37344) on Carbachol and Efs–Evoked Contractions of Isolated Ovine Detrusor Muscle Strip

Preventive Effect of Hydrogen Water on the Development of Detrusor Overactivity in a Rat Model of Bladder Outlet Obstruction

Bladder ischemia and oxidative stress contribute to the pathogenesis of bladder dysfunction caused by bladder outlet obstruction. H2 reportedly acts as an effective antioxidant. We investigated whether oral ingestion of H2 water would have a beneficial effect on bladder function in a rat model of bladder outlet obstruction. H2 water was made by dissolving H2 gas in ordinary drinking water using a hydrogen water producing apparatus. The bladder outlet obstruction model was surgically induced in male rats. Rats with obstruction were fed H2 water or ordinary drinking water. On week 4 postoperatively cystometry was performed. Oxidative stress markers and the bladder nerve growth factor level were determined. Bladder tissues were processed for pharmacological studies and histological analysis. The micturition interval and micturition volume significantly decreased in obstructed rats given ordinary drinking water. These decreases were significantly suppressed by oral ingestion of H2 water. Increased post-void residual volume in obstructed rats was significantly reduced by H2 water. Obstruction led to a significant increase in bladder weight, oxidative stress markers and nerve growth factor. H2 water significantly suppressed these increases without affecting bladder weight. There was no significant difference in histological findings between rats with bladder obstruction given H2 water and ordinary drinking water. Decreased responses of detrusor muscle strips from obstructed bladders to KCl, carbachol and electrical field stimulation were reversed by H2 water ingestion. Results suggest that H2 water could ameliorate bladder dysfunction secondary to bladder outlet obstruction by attenuating oxidative stress.

The role of muscarinic receptor subtypes on carbachol-induced contraction of normal human detrusor and overactive detrusor associated with benign prostatic hyperplasia

The aim of this study was to compare the effect of antimuscarinic antagonists on carbachol-induced contraction of normal human bladder and detrusor overactivity associated with benign prostatic hyperplasia (DO/BPH). Samples of human bladder muscle were obtained from patients undergoing total cystectomy for bladder cancer (normal bladder), and those undergoing retropubic prostatectomy for BPH. All of the patients with DO/BPH had detrusor overactivity according to urodynamic studies. Detrusor muscle strips were mounted in 10-ml organ baths containing Krebs solution, and concentration–response curves for carbachol were obtained in the presence of antimuscarinic antagonists (4-DAMP, methoctramine, pirenzepine, tolterodine, solifenacin, trospium, propiverine, oxybutynin, and imidafenacin) or vehicle. All antagonists competitively antagonized concentration–response curves to carbachol with high affinities in normal bladder. The rank order of mean pA2 values was as follows: trospium (10.1) > 4-DAMP (9.87), imidafenacin (9.3) > solifenacin (8.8) > tolterodine (8.6) > oxybutynin (8.3) > propiverine (7.7) > pirenzepine (7.4) > methoctramine (6.6). The effects of these antimuscarinic antagonists did not change when tested with DO/BPH bladder, suggesting that each antimuscarinic antagonist has a similar effect in this condition. Schild plots showed a slope corresponding to unity, except for propiverine with DO/BPH detrusor. In conclusion, M3-receptors mainly mediate contractions in human bladder strips with normal state and DO/BPH.

Mirodenafil prevents bladder dysfunction induced by chronic bladder ischemia in rats.

Purpose:To investigate the protective effect of mirodenafil on bladder function in a rat model of chronic bladder ischemia (CBI).Methods:Twenty-four Sprague-Dawley rats were randomized to three groups: untreated, sham-operated rats (control group); untreated, CBI model rats (CBI group); and CBI rats treated daily with 4 mg/kg mirodenafil (CBI+mirodenafil group). The CBI and CBI+mirodenafil groups underwent endothelial injury to the iliac arteries and were fed a 2% cholesterol diet after injury. Four weeks after surgery, the CBI+mirodenafil group started daily treatment with mirodenafil for four weeks. Eight weeks after surgery, continuous in vivo cystometry and in vivo organ bath studies of detrusor muscle strips were performed.Results:in vivo cystometry revealed that the rats in the CBI group had a significantly higher micturition frequency, lower bladder capacity, and lower compliance than the rats in the control and CBI+mirodenafil groups. The detrusor muscle strip study showed that the magnitude of the carbachol-induced contractile response was significantly lower in the CBI group compared to either the control or CBI+mirodenafil group. Addition of daily mirodenafil after induction of CBI decreased the contractile response, compared to untreated CBI rats. CBI induced submucosal fibrosis and degenerative changes in bladder walls, which was reversed by the addition of mirodenafil.Conclusions:Daily treatment with mirodenafil showed protective effects against bladder dysfunction resulting from CBI in rats.