Detrusor Smooth Muscle Contractility Research Articles

Human HPSE2 gene transfer ameliorates bladder pathophysiology in a mutant mouse model of urofacial syndrome.

Rare early-onset lower urinary tract disorders include defects of functional maturation of the bladder. Current treatments do not target the primary pathobiology of these diseases. Some have a monogenic basis, such as urofacial, or Ochoa, syndrome (UFS). Here, the bladder does not empty fully because of incomplete relaxation of its outflow tract, and subsequent urosepsis can cause kidney failure. UFS is associated with biallelic variants of HPSE2, encoding heparanase-2. This protein is detected in pelvic ganglia, autonomic relay stations that innervate the bladder and control voiding. Bladder outflow tracts of Hpse2 mutant mice display impaired neurogenic relaxation. We hypothesized that HPSE2 gene transfer soon after birth would ameliorate this defect and explored an adeno-associated viral (AAV) vector-based approach. AAV9/HPSE2, carrying human HPSE2 driven by CAG, was administered intravenously into neonatal mice. In the third postnatal week, transgene transduction and expression were sought, and ex vivo myography was undertaken to measure bladder function. In mice administered AAV9/HPSE2, the viral genome was detected in pelvic ganglia. Human HPSE2 was expressed and heparanase-2 became detectable in pelvic ganglia of treated mutant mice. On autopsy, wild-type mice had empty bladders, whereas bladders were uniformly distended in mutant mice, a defect ameliorated by AAV9/HPSE2 treatment. Therapeutically, AAV9/HPSE2 significantly ameliorated impaired neurogenic relaxation of Hpse2 mutant bladder outflow tracts. Impaired neurogenic contractility of mutant detrusor smooth muscle was also significantly improved. These results constitute first steps towards curing UFS, a clinically devastating genetic disease featuring a bladder autonomic neuropathy.

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 ...

SKA-31-induced activation of small-conductance calcium-activated potassium channels decreased modulation of detrusor smooth muscle function in a rat model of obesity

Increased excitability and contractility of detrusor smooth muscle (DSM) cells are associated with overactive bladder (OAB), which is often induced by obesity. Small-conductance Ca2+-activated K+ (SK) channels regulate the excitability and contractility of DSM cells. Selective pharmacological activation of SK channels attenuates hyperpolarization and the decreased relaxation effect in DSM cells in obesity-induced OAB. However, additional data are needed to confirm the regulatory effect of SK channels on the function of DSM cells in obesity-related OAB. The tested hypothesis was that activation of SK channels decreases modulation of DSM function in a rat model of obesity-related OAB. Female Sprague Dawley rats were fed a normal diet (ND) or a high-fat diet (HFD), weighed after 12weeks, and subjected to urodynamic study, patch-clamp electrophysiology, and isometric tension recording. The average body weight and incidence of OAB were increased in the HFD group. Patch-clamp studies revealed that pharmacological activation of SK channels with SKA-31 had attenuated hyperpolarization of DSM cells. In addition, isometric tension recordings indicated that SKA-31 decreased relaxation of spontaneous phasic contractions of DSM strips in the HFD group. Attenuated function of SK channels increased the excitability and contractility of DSM cells, which contributed to the occurrence of OAB, suggesting that SK channels are potential therapeutic targets for control of OAB.

The inhibitory effect of trimetazidine on detrusor contractility - a potential repositioning of trimetazidine for the treatment of overactive bladder.

This study aimed to identify the effect of trimetazidine (TMZ), an antianginal drug, on detrusor smooth muscle (DSM) contractility and its possible mechanisms of action. We performed in-vitro contractility studies on isolated mouse DSM strips and investigated the effect of TMZ on Ca2+ levels in fura-2-loaded A7r5 cells. TMZ (300 or 1000 µM) inhibited carbachol (CCh)- and KCl-induced contractions and produced a concentration-dependent (10-1000 µM) relaxation in KCl-precontracted DSM strips. TMZ-induced relaxation was markedly decreased by BaCl2, an inward-rectifying K+ channel blocker, but was not altered by preincubation with tetraethylammonium, glibenclamide, 4-aminopyridine, propranolol, L-NAME or methylene blue. TMZ (300 or 1000 µM) reduced both the CaCl2-induced contraction of depolarized DSM strips under Ca2+-free conditions and the CCh-induced contraction of DSM strips preincubated with nifedipine in Ca2+-containing Krebs solution. Furthermore, TMZ (1000 µM) significantly decreased the Ca2+ levels in fura-2-loaded A7r5 cells. TMZ decreased DSM contractility and caused a concentration-dependent relaxation of the tissue possibly through its actions on Ca2+ transients and K+ channels. Our results provide preclinical evidence that TMZ would be a potential candidate to treat disorders related to the overactivity of the bladder.

Alterations in detrusor contractility in rat model of bladder cancer

Urinary incontinence of idiopathic nature is a common complication of bladder cancer, yet, the mechanisms underlying changes in bladder contractility associated with cancer are not known. Here by using tensiometry on detrusor smooth muscle (DSM) strips from normal rats and rats with bladder cancer induced by known urothelial carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), we show that bladder cancer is associated with considerable changes in DSM contractility. These changes include: (1) decrease in the amplitude and frequency of spontaneous contractions, consistent with the decline of luminal pressures during filling, and detrusor underactivity; (2) diminution of parasympathetic DSM stimulation mainly at the expense of m-cholinergic excitatory transmission, suggestive of difficulty in bladder emptying and weakening of urine stream; (3) strengthening of TRPV1-dependent afferent limb of micturition reflex and TRPV1-mediated local contractility, promoting urge incontinence; (4) attenuation of stretch-dependent, TRPV4-mediated spontaneous contractility leading to overflow incontinence. These changes are consistent with the symptomatic of bladder dysfunction in bladder cancer patients. Considering that BBN-induced urothelial lesions in rodents largely resemble human urothelial lesions at least in their morphology, our studies establish for the first time underlying reasons for bladder dysfunction in bladder cancer.

New mouse model of underactive bladder developed by placement of a metal ring around the bladder neck.

To develop a new mouse model of underactive bladder (UAB) caused by chronic bladder outlet obstruction (BOO). BOO was created in 6-week-old male C57BL/6 mice using surgery to loosely place a silver jump ring around the bladder neck of each mouse. Micturition behavior (assessed with a metabolic cage) and cystometry were used to evaluate bladder function at 8 and 16 weeks after BOO. Following completion of the functional studies, the bladders of the mice were excised, weighed, and subjected to histological analysis. Micturition behavior analysis showed that mice subjected to BOO for 16 weeks had a lower frequency of micturition (7.3 ± 1.1 vs 12.5 ± 3.0 times/d, P < .05) and volume per void (106.0 ± 0.1 vs 133.9 ± 3.2 μL, P < .05) than mice subjected to BOO for 8 weeks. Cystometry revealed that mice subjected to BOO for 16 weeks had lower baseline pressure (8.4 ± 0.6 vs 14.0 ± 0.7 cmH2 O, P < .01) and micturition pressure (13.9 ± 1.1 vs 42.8 ± 1.7 cmH2 O, P < .05) than mice subjected to BOO for 8 weeks. BOO caused progressive increases in bladder mass and collagen deposition over time. We successfully established a novel mouse model of UAB using surgery to place a silver jump ring loosely on the bladder neck. BOO initially induced bladder overactivity but subsequently resulted in UAB due to deterioration of detrusor smooth muscle contractility and progressive deposition of collagen in the bladder wall.

Comparative effects of angiotensin II on the contractility of muscularis mucosae and detrusor in the pig urinary bladder.

To explore contractile actions of angiotensin II (ATII) on the muscularis mucosae (MM) of the bladder, ATII-induced contractions were compared between MM and the detrusor smooth muscle (DSM) of the pig bladder by isometric tension recordings. Effects of ATII on spontaneous Ca2+ transients in MM were visualized using Cal-520 fluorescence. ATII receptor type 1 (ATR1) expression in MM and DSM was also examined by immunohistochemistry. ATII (1 nM-1 μM) caused phasic contractions of MM in a concentration-dependent manner, while ATII (10 nM-10 μM) had no or marginal effects on DSM contractility. ATII (100 nM)-induced MM contractions had an amplitude of approximately 70% of carbachol (1 μM)-induced or 90% of U46619 (100 nM)-induced contractions. Candesartan (10 nM), an ATR1 blocker, prevented the contractile effects of ATII (1 nM) in MM, while ATR1 immunofluorescence was greater in MM than DSM. ATII (10-100 pM) increased the frequency but not the amplitude of spontaneous Ca2+ transients in MM. Both urothelium-intact and -denuded MM strips developed comparable spontaneous phasic contractions, but ATII, carbachol and U46619-induced contractions were significantly larger in urothelium-denuded than urothelium-intact MM strips. In conclusion, the MM appears to have a much greater sensitivity to ATII compared with DSM that could well sense circulating ATII, suggesting that MM may be the predominant target of contractile actions induced by ATII in the bladder while the urothelium appears to inhibit MM contractility.

Detrusor Smooth Muscle KV7 Channels: Emerging New Regulators of Urinary Bladder Function.

Relaxation and contraction of the urinary bladder smooth muscle, also known as the detrusor smooth muscle (DSM), facilitate the micturition cycle. DSM contractility depends on cell excitability, which is established by the synchronized activity of multiple diverse ion channels. K+ channels, the largest family of channels, control DSM excitability by maintaining the resting membrane potential and shaping the action potentials that cause the phasic contractions. Among the members of the voltage-gated K+ (KV) channel superfamily, KV type 7 (KV7) channels — KV7.1–KV7.5 members encoded by KCNQ1–KCNQ5 genes — have been recently identified as functional regulators in various cell types including vascular, cardiac, and neuronal cells. Their regulatory roles in DSM, however, are just now emerging and remain to be elucidated. To address this gap, our research group has initiated the systematic investigation of human DSM KV7 channels in collaboration with clinical urologists. In this comprehensive review, we summarize the current understanding of DSM Kv7 channels and highlight recent discoveries in the field. We describe KV7 channel expression profiles at the mRNA and protein levels, and further elaborate on functional effects of KV7 channel selective modulators on DSM excitability, contractility, and intracellular Ca2+ dynamics in animal species along with in vivo studies and the limited data on human DSM. Within each topic, we highlight the main observations, current gaps in knowledge, and most pressing questions and concepts in need of resolution. We emphasize the lack of systematic studies on human DSM KV7 channels that are now actively ongoing in our laboratory.

TRPM4 channel inhibitors 9-phenanthrol and glibenclamide differentially decrease guinea pig detrusor smooth muscle whole-cell cation currents and phasic contractions.

Nonselective cation channels, consistent with transient receptor potential melastatin-4 (TRPM4), regulate detrusor smooth muscle (DSM) function. TRPM4 channels can exist as homomers or assemble with sulfonylurea receptors (SURs) as complexes. We evaluated contributions of TRPM4/SUR-TRPM4 channels to DSM excitability and contractility by examining the effects of TRPM4/SUR-TRPM4 channel modulators 9-phenanthrol, glibenclamide, and diazoxide on freshly-isolated guinea pig DSM cells (amphotericin-B perforated patch-clamp electrophysiology) and mucosa-free DSM strips (isometric tension recordings). In DSM cells, complete removal of extracellular Na+ decreased voltage-step-induced cation (non-K+ selective) currents. At high positive membrane potentials, 9-phenanthrol at 100 μM attenuated voltage step-induced currents more effectively than at 30 μM, revealing concentration-dependent, voltage-sensitive inhibition. In comparison to 9-phenanthrol, glibenclamide (100 μM) displayed lower inhibition of cation currents. In the presence of glibenclamide (100 μM), 9-phenanthrol (100 μM) further decreased the currents. The SUR-TRPM4 complex activator diazoxide (100-300 μM) weakly inhibited the currents. 9-Phenanthrol, but not glibenclamide or diazoxide, increased cell capacitance (a cell surface area indicator). In contractility studies, glibenclamide displayed lower potencies than 9-phenanthrol attenuating spontaneous and 20 mM KCl-induced DSM phasic contractions. While both compounds showed similar maximum inhibitions on DSM spontaneous phasic contractions, glibenclamide was generally less efficacious on 20 mM KCl-induced phasic contractions. In summary, the observed differential effects of 9-phenanthrol and glibenclamide on DSM excitability and contractility support unique mechanisms for the two compounds. The data suggest that SUR-TRPM4 complexes do not contribute to DSM function. This study advances our understanding of pharmacological effects of glibenclamide and 9-phenanthrol on DSM cell cation currents.

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.

Impairment of cholinergic bladder contractility in rat model of type I diabetes complicated by cystitis: Contribution of neurotransmitter-degrading ectoenzymes

Parasympathetic regulation of urinary bladder contractions primarily involves acetylcholine release and activation of detrusor smooth muscle (DSM) muscarinic acetylcholine (mACh) receptors. Co-release of ATP and activation of DSM purinergic P2X1-receptors may participate as well in some species. Both types of neuromuscular transmission (NMT) are impaired in diabetes, however, which factors may contribute to such impairment remains poorly understood. Here by using rats with streptozotocin(STZ)-induced type I diabetes (8th week after induction) we show that contribution of atropine-sensitive m-cholinergic component to the contractions of urothelium-denuded DSM strips evoked by electric field stimulation (EFS) greatly increased when diabetic bladders presented overt signs of accompanying cystitis. Modeling of hemorrhagic cystitis alone in control rats by cyclophosphamide injection only modestly increased m-cholinergic component of EFS-contractions. However, exposure of DSM strips from control animals to acetylcholinesterase (AChE) inhibitor, neostigmine (1–10 μM) largely reproduced alterations in EFS contractions observed in diabetic DSM complicated by cystitis. Ellman's assay revealed statistically significant 31% decrease of AChE activities in diabetic vs. control DSM. Changes in purinergic contractility of diabetic DSM were consistent with altered P2X1-receptor desensitization and re-sensitization. They could be mimicked by pharmacological inhibition of ATP-degrading ecto-ATPases with ARL 67156 (50 μM), pointing to compromised extracellular ATP clearance as underlying reason. We conclude that decreased AChE activities associated with diabetes and likely cystitis provide complementary factor to the described in literature altered expression of mACh receptor subtypes linked to diabetes as well as to cystitis to produce dramatic modification of cholinergic NMT.

Doxorubicin induces detrusor smooth muscle impairments through myosin dysregulation, leading to a risk of lower urinary tract dysfunction.

Cytotoxic chemotherapy is the foundation for the treatment of the wide variety of childhood malignancies; however, these therapies are known to have a variety of deleterious side effects. One common chemotherapy used in children, doxorubicin (DOX), is well known to cause cardiotoxicity and cardiomyopathy. Recent studies have revealed that DOX impairs skeletal and smooth muscle function and contributes to fatigue and abnormal intestinal motility in patients. In this study, we tested the hypothesis that systemic DOX administration also affects detrusor smooth muscle (DSM) function in the urinary bladder, especially when administered at a young age. The effects on the DSM and bladder function were assessed in BALB/cJ mice that received six weekly intravenous injections of DOX (3 mg·kg-1·wk-1) or saline for the control group. Systemic DOX administration resulted in DSM hypertrophy, increased voiding frequency, and a significant attenuation of DSM contractility, followed by a slower relaxation compared with the control group. Gene expression analyses revealed that unlike DOX-induced cardiotoxicity, the bladders from DOX-administered animals showed no changes in oxidative stress markers; instead, downregulation of large-conductance Ca2+-activated K+ channels and altered expression of myosin light-chain kinase coincided with reduced myosin light-chain phosphorylation. These results indicate that in vivo DOX exposure caused DSM dysfunction by dysregulation of molecules involved in the detrusor contractile-relaxation mechanisms. Collectively, our findings suggest that survivors of childhood cancer treated with DOX may be at increased risk of bladder dysfunction and benefit from followup surveillance of bladder function.

Compound‐dependent Effects of TRPM4 Channel Modulators on Guinea Pig Detrusor Smooth Muscle Excitability and Contractility

Guinea pig detrusor smooth muscle (DSM) cells express transient receptor potential melastatin 4 (TRPM4) channels, proposed to regulate DSM excitability and contractility1. Supporting evidence with the TRPM4 channel inhibitor 9‐phenanthrol showed reduced guinea pig DSM phasic contractions, and induced DSM cell hyperpolarization and decreased voltage‐step evoked cation currents1. Recently, a concern has been raised about selectivity of 9‐phenanthrol2–4. Here, we used whole‐cell amphotericin‐B perforated and inside‐out single‐channel patch‐clamp techniques, and isometric DSM tension recordings to address the hypothesis that TRPM4 channels regulate DSM excitation‐contraction coupling by examining the novel selective TRPM4 channel inhibitor 4‐chloro‐2‐[2‐(2‐chloro‐phenoxy)‐acetylamino]‐benzoic acid (CBA, compound 5 in reference5), along with non‐selective TRPM4 channel modulators: flufenamic acid (FFA)5 and BTP2 (or YM‐58483)6, a blocker and an activator, respectively. DSM cell voltage‐step induced cation currents were not increased when held at +26 or +6 mV in‐between voltage steps in comparison to the standard −74 mV (n=7), inconsistent with presence of TRPM4 currents6. Separate applications of CBA (30 μM) or BTP2 (10 μM) in DSM cells did not change the voltage‐step evoked currents (n=5–6). In contrast, FFA (100 μM) caused an increase at positive voltages, 1.4‐fold at +106 mV (p&lt;0.05, n=7). In the same DSM cells, 9‐phenanthrol (100 μM) added following washout of FFA or in the presence of either CBA or BTP2 reduced the currents, 42–57% at +106 mV (p&lt;0.05, n=4–7). 9‐Phenanthrol but not CBA, FFA, or BTP2 altered cell capacitance, 1.1‐fold increase (p&lt;0.05). Excised single‐channel recordings revealed a non‐TRPM4 channel cation conductance of ~15 pS that was blocked by 9‐phenanthrol (30 μM). CBA, added cumulatively up to 100 μM, showed either no or very weak effects (maximum inhibitions: 8–38%) on spontaneous and 20 mM KCl‐induced phasic contraction parameters (n=10–13). For 300 μM CBA, inhibitions were higher (up to 68%) on all contraction parameters except for the duration of KCl‐induced contractions, which increased 2‐fold (n=7–9). 9‐Phenanthrol examined in parallel showed robust reductions in DSM phasic contraction parameters (IC50 values: 1.1–21 μM and maximum inhibitions: 35–86%, n=11–20). In summary, 9‐phenanthrol ‐ in contrast to other tested TRPM4 channel modulators: CBA, FFA, or BTP2 ‐effectively reduced DSM cell voltage‐step induced cation currents and DSM strip contractions. Our results reveal differential effects of TRPM4 channel modulators on guinea pig DSM excitability and contractility. Future studies are needed to determine the underlying mechanisms and to elucidate the function of TRPM4 in the urinary bladder. Interpretation of the results with 9‐phenanthrol requires caution especially related to its perceived TRPM4 channel selectivity.Support or Funding InformationNIH DK106964 to Georgi V. PetkovThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of high-fat diet-induced obesity on the small-conductance Ca2+-activated K+ channel function affecting the contractility of rat detrusor smooth muscle.

Obesity usually induces overactive bladder (OAB) associated with detrusor overactivity, which is related to increased contractility of the detrusor smooth muscle (DSM). Small-conductance Ca2+-activated K+ (SK) channels play a constitutive role in the regulation of DSM contractility. However, the role of SK channels in the DSM changes in obesity-related OAB is still unknown. Here, we tested the hypothesis that obesity-related OAB is associated with reduced expression and activity of SK channels in DSM and that SK channels activation is a potential treatment for OAB. Female Sprague-Dawley rats were fed a normal diet (ND) or a high-fat diet (HFD) and weighed after 12 weeks. Urodynamic studies, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and isometric tension recording were performed. Increased average body weights and urodynamically demonstrated OAB were observed in HFD rats. qRT-PCR experiments revealed a decrease in the mRNA expression level of SK channel in DSM tissue of the HFD rats. Isometric tension recordings indicated an attenuated relaxation effect of NS309 on the spontaneous phasic and electrical field stimulation-induced contractions that occurred via SK channel activation in HFD DSM strips. Reduced expression and activity of SK channels in the DSM contribute to obesity-related OAB, indicating that SK channels are a potential therapeutic target for OAB.

Chloride Single Channel Activity in Freshly‐Isolated Guinea Pig Detrusor Smooth Muscle Cells

Ion channels in detrusor smooth muscle (DSM) control excitability and contractility of the urinary bladder. Several ion channel types have already been detected and characterized in DSM cells, especially voltage‐gated cationic channels. However, little is known about Cl− channels in DSM cells. Specifically, it is unknown what Cl− channel subtypes are expressed in the plasma membrane of DSM cells and how they control DSM cell excitability. Earlier molecular and fluorometric studies suggested the presence of Ca2+‐activated Cl− channels in DSM cells. Our Agilent Human GE 4x44K v2 microarray data analysis on single DSM cell mRNA detected a number of candidates underlying Cl− conductance in DSM cells including plasmalemmal TMEM16A/ANO1, CLCN3, CLCN6, and CLCN7. Thus, detailed biophysical analyses are needed to characterize these Cl− channels. Here, guinea pig DSM cells were freshly isolated by an enzymatic digestion method with papain and collagenase type II, plated at the glass bottom of a recording chamber, and electrophysiologically tested within 6 hours after the isolation. Inside‐out single channel patch clamp recording technique was used to detect electrical activity of Clchannels in the plasma membrane of isolated DSM cells. To separate Cl− from cationic currents, all experiments were done using asymmetrical Cl− and symmetrical Na+ solutions. The bath/intracellular solution contained (in mM): 110 Na‐glutamate, 5 NaCl, 60 mannitol, 10 HEPES, 1 CaCl2, 1 CoCl2, 0.0005 phorbol 12‐myristate 13‐acetate, pH 7.2; the pipette/extracellular contained (in mM): 110 NaCl, 1.5 MgCl2, 2 CaCl2, 60 mannitol, 10 HEPES, 0.001 paxilline, 0.01 nifedipine, pH 7.4. Cl− single channel activity detection frequency varied from zero to 26.7 % within a single DSM cell preparation with an average frequency for detection of 7.4 % (13 out of 175 patches, 8 guinea pigs). Cl− channels displayed unique gating with multiple subconductive states and a fully opened single‐channel conductance in a range from 140 pS to 193 pS with an average value of 163 ± 15 pS (12 patches, 8 guinea pigs, mean ± SD). A linear fitting of single channel current amplitude – membrane potential relationships yielded a reversal potential of − 41.3 ± 5.5 mV (12 patches, 8 guinea pigs, mean ± SD) for this Cl− channel, close to the theoretical ECl of − 65 mV and confirming that this channel is permeable to Cl−. The Cl− channel demonstrated strong voltage‐dependence of activation with virtually no activity below the measured reversal potential and always robust prolonged openings at depolarizing voltages. The channel had similar gating when exposed to the intracellular Ca2+‐free (buffered with 5 mM EGTA in nominal Ca2+) and 1 mM Ca2+ solutions. The Cl− channel was not blocked by 9‐phenanthrol (30 μM), a transient receptor potential melastatin 4 channel inhibitor that also has been reported to block the activity of TMEM16A Ca2+‐activated Cl− channels in vascular smooth muscle cells. In conclusion, our data reveal for the first time the presence of Ca2+‐insensitive voltage‐dependent Cl− channel in guinea pig DSM cells at the single channel level. This channel may be a key regulator of DSM excitability.Support or Funding InformationSupported by NIH R01DK106964 to Georgi V. Petkov.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

KV7 Channel Pharmacological Activation by the Novel Activator ML213: Role for Heteromeric KV7.4/KV7.5 Channels in Guinea Pig Detrusor Smooth Muscle Function.

Voltage-gated KV7 channels (KV7.1 to KV7.5) are important regulators of the cell membrane potential in detrusor smooth muscle (DSM) of the urinary bladder. This study sought to further the current knowledge of KV7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca2+ imaging, amphotericin-B perforated patch-clamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound N-(2,4,6-trimethylphenyl)-bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of KV7.2, KV7.4, and KV7.5 channels, to examine their physiologic roles in guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 µM) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the KV7 channel inhibitor XE991 (10 µM). ML213 (0.1-30 µM) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 µM) decreased global intracellular Ca2+ concentrations in Fura-2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 µM) caused an increase in the amplitude of whole-cell KV7 currents. Further, in current-clamp mode of the perforated patch clamp, ML213 hyperpolarized DSM cell membrane potential in a manner reversible by washout or XE991 (10 µM), consistent with ML213 activation of KV7 channel currents. Preapplication of XE991 (10 µM) not only depolarized the DSM cells, but also blocked ML213-induced hyperpolarization, confirming ML213 selectivity for KV7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric KV7.4/KV7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive KV7.4- and KV7.5-containing channels are essential regulators of DSM excitability and contractility.

Preventative effects of a HIF inhibitor, 17-DMAG, on partial bladder outlet obstruction-induced bladder dysfunction.

Posterior urethral valves are the most common cause of partial bladder outlet obstruction (PBOO) in the pediatric population. Pathological changes in the bladder developed during PBOO are responsible for long-lasting voiding dysfunction in this population despite early surgical interventions. Increasing evidence showed PBOO induces an upregulation of hypoxia-inducible factors (HIFs) and their transcriptional target genes, and they play a role in pathophysiological changes in the obstructed bladders. We hypothesized that blocking HIF pathways can prevent PBOO-induced bladder dysfunction. PBOO was surgically created by ligation of the bladder neck in male C57BL/6J mice for 2 wk. PBOO mice received intraperitoneal injection of either saline or 17-DMAG (alvespimycin, 3 mg/kg) every 48 h starting from day 1 postsurgery. Sham-operated animals received injection of saline on the same schedule as PBOO mice and served as controls. The bladders were harvested after 2 wk, and basal activity and evoked contractility of the detrusor smooth muscle (DSM) were evaluated in vitro. Bladder function was assessed in vivo by void spot assay and cystometry in conscious, unrestrained mice. Results indicated the 17-DMAG treatment preserved DSM contractility and partially prevented the development of detrusor over activity in obstructed bladders. In addition, PBOO caused a significant increase in the frequency of micturition, which was significantly reduced by 17-DMAG treatment. The 17-DMAG treatment improved urodynamic parameters, including increases in the bladder pressure at micturition and nonvoid contractions observed in PBOO mice. These results demonstrate that treatment with 17-DMAG, a HIF inhibitor, significantly alleviated PBOO-induced bladder pathology in vivo.

Nongenomic modulation of the large conductance voltage- and Ca2+-activated K+ channels by estrogen: A novel regulatory mechanism in human detrusor smooth muscle.

Estrogens have an important role in regulating detrusor smooth muscle (DSM) function. However, the underlying molecular and cellular mechanisms by which estrogens control human DSM excitability and contractility are not well known. Here, we used human DSM specimens from open bladder surgeries on 27 patients to elucidate the mechanism by which 17β‐estradiol regulates large conductance voltage‐ and Ca2+‐activated K+ (BK) channels, the most prominent K+ channels in human DSM. We employed single BK channel recordings on inside‐out excised membrane patches, perforated whole‐cell patch‐clamp on freshly isolated DSM cells, and isometric tension recordings on DSM‐isolated strips to investigate the mechanism by which 17β‐estradiol activates BK channels. 17β‐Estradiol (100 nmol/L) rapidly increased depolarization‐induced whole‐cell K+ currents in DSM cells. The 17β‐estradiol stimulatory effects on whole‐cell BK currents were completely abolished by the selective BK channel inhibitor paxilline (1 μmol/L), clearly indicating that 17β‐estradiol specifically activates BK channels. 17β‐Estradiol also increased the frequency of ryanodine receptor‐mediated transient BK currents. Single BK channel recordings showed that 17β‐estradiol (100 nmol/L) significantly increased the BK channel open probability of inside‐out excised membrane patches, revealing that 17β‐estradiol activates BK channels directly. 17β‐Estradiol reduced spontaneous phasic contractions of human DSM‐isolated strips in a concentration‐dependent manner (100 nmol/L‐1 μmol/L), and this effect was blocked by paxilline (1 μmol/L). 17β‐Estradiol (100 nmol/L) also reduced nerve‐evoked contractions of human DSM‐isolated strips. Collectively, our results reveal that 17β‐estradiol plays a critical role in regulating human DSM function through a direct nongenomic activation of BK channels.

AB317. SPR-44 Pharmacological activation of individual KCNQ channel subtypes in detrusor smooth muscle represents a promising novel approach for overactive bladder treatment

ObjectiveOur recent studies have demonstrated voltage-gated KCNQ channels (KCNQ1-KCNQ5) as key regulators of detrusor smooth muscle (DSM) function. Despite emerging developments, the physiological role of individual KCNQ channel subtypes remains less clear. Here, we utilized the novel compound ML-213, a potent activator of KCNQ2, KCNQ4, and KCNQ5 channels, to elucidate their physiological roles in guinea pig DSM function.MethodsUsing isometric DSM tension recordings, Ca2+ imaging, and amphotericin-B perforated patch-clamp electrophysiology, we elucidated the role of ML-213-sensitive KCNQ channels in regulating DSM excitability and contractility.ResultsML-213 concentration-dependently (100 nM–30 µM) inhibited spontaneous phasic, pharmacologically-induced, and nerve-evoked contractions in DSM isolated strips. ML-213 (10 µM) decreased the global intracellular Ca2+ concentrations in DSM isolated strips, effects blocked by the L-type voltage-gated Ca2+ (CaV) channel inhibitor nifedipine (1 µM) and the KCNQ1-KCNQ5 channel inhibitor XE991 (10 µM). These data suggest that ML-213 decreases the global intracellular Ca2+ concentration by inhibiting L-type CaV channels through an indirect mechanism downstream from KCNQ channel activation. In addition, ML-213 hyperpolarized the cell membrane potential and inhibited spontaneous action potentials in DSM cells, effects reversible by washout. We next aimed to examine the effects of ML-213 on whole cell KCNQ currents. To isolate KCNQ currents, the bath solution contained the large conductance voltage- and Ca2+-activated K+ channel inhibitor paxilline (1 µM) and gadolinium chloride (GdCl3, 50 µM), which blocks L-type CaV channels and non-selective cation channels. Under these experimental conditions, ML-213 (10 µM) enhanced whole cell KCNQ currents. These findings suggest that the modulation of K+ transport through ML-213-sensitive KCNQ channels underlies ML-213-induced cell membrane hyperpolarization to decrease the global intracellular Ca2+ concentration and DSM contractility.ConclusionsThese data using the novel compound ML-213, suggest that KCNQ2-, KCNQ4-, and KCNQ5-containing channels are essential regulators of the excitability, intracellular Ca2+ concentration, and contractility of DSM by virtue of their control of the membrane potential. Moreover, these new findings provide a foundational basis for future investigations on KCNQ channel functional roles in human DSM excitability and contractility to confirm their potential as novel therapeutic targets for overactive bladder.Source of FundingNIH grant R01-DK106964 to GV Petkov and F31-DK104528 to A Provence.