Detrusor Smooth Muscle Research Articles

Spontaneous synaptic drive in detrusor smooth muscle: computational investigation and implications for urinary bladder function.

The detrusor, a key component of the urinary bladder wall, is a densely innervated syncytial smooth muscle tissue. Random spontaneous release of neurotransmitter at neuromuscular junctions (NMJs) in the detrusor gives rise to spontaneous excitatory junction potentials (SEJPs). These sub-threshold passive signals not only offer insights into the syncytial nature of the tissue, their spatio-temporal integration is critical to the generation of spontaneous neurogenic action potentials which lead to focal contractions during the filling phase of the bladder. Given the structural complexity and the contractile nature of the tissue, electrophysiological investigations on spatio-temporal integration of SEJPs in the detrusor are technically challenging. Here we report a biophysically constrained computational model of a detrusor syncytium overlaid with spatially distributed innervation, using which we explored salient features of the integration of SEJPs in the tissue and the key factors that contribute to this integration. We validated our model against experimental data, ascertaining that observations were congruent with theoretical predictions. With the help of comparative studies, we propose that the amplitude of the spatio-temporally integrated SEJP is most sensitive to the inter-cellular coupling strength in the detrusor, while frequency of observed events depends more strongly on innervation density. An experimentally testable prediction arising from our study is that spontaneous release frequency of neurotransmitter may be implicated in the generation of detrusor overactivity. Set against histological observations, we also conjecture possible changes in the electrical activity of the detrusor during pathology involving patchy denervation. Our model thus provides a physiologically realistic, heuristic framework to investigate the spread and integration of passive potentials in an innervated syncytial tissue under normal conditions and in pathophysiology.

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.

ATP transients accompany spontaneous contractions in isolated guinea-pig detrusor smooth muscle.

What is the central question of this study? Overactive bladder is associated with enhanced spontaneous contractions, but their origins are unclear. The aim of this study was to characterize the accompanying ATP transients. What is the main finding and its importance? Spontaneous detrusor contractions were accompanied by transient increases of ATP, and their appearance was delayed by previous activation of efferent nerves to the detrusor. This indicates that spontaneous ATP release from nerve terminals supports spontaneous contractions. ATP is a functional excitatory neurotransmitter in human bladder only in pathologies such as overactive bladder. A potential drug target is revealed to manage this condition. Spontaneous contractions are characteristic of the bladder wall, but their origins remain unclear. Activity is reduced if the mucosa is removed but does not disappear, suggesting that a fraction arises from the detrusor. We tested the hypothesis that spontaneous detrusor contractions arise from spontaneous ATP release. Guinea-pig detrusor strips, without mucosa, were superfused with Tyrode solution at 36°C. Preparations were subjected to electrical field stimulation (EFS; 3s trains at 90s intervals) to produce nerve-mediated contractions, abolished by 1µm TTX. Amperometric ATP electrodes on the preparation surface recorded any ATP released. Spontaneous contractions and ATP transients were recorded between EFS trains. Nerve-mediated contractions were attenuated by atropine and α,β-methylene ATP; in combination, they nearly abolished contractions, as did nifedipine. Contractions were accompanied by ATP transients that were unaffected by atropine but inhibited by TTX and greatly attenuated by nifedipine. Spontaneous contractions were accompanied by ATP transients, with a close correlation between the magnitudes of both transients. ATP and contractile transients persisted with TTX, atropine and nifedipine. Immediately after a nerve-mediated contraction and ATP transient, there was a longer interval than normal before spontaneous activity resumed. Spontaneous contractions and ATP transients are proposed to arise from ATP leakage from nerve terminals innervating the detrusor. Extracellular ATP has a greater functional significance in humans who suffer from detrusor overactivity (spontaneous bladder contractions associated with incontinence) owing to its reduced hydrolysis at the nerve-muscle interface. This study shows the origin of spontaneous activity that might be exploited to develop a therapeutic management of this condition.

Extracellular pH and intracellular phosphatidylinositol 4,5-bisphosphate control Cl- currents in guinea pig detrusor smooth muscle cells.

Cl- channels serve as key regulators of excitability and contractility in vascular, intestinal, and airway smooth muscle cells. We recently reported a Cl- conductance in detrusor smooth muscle (DSM) cells. Here, we used the whole cell patch-clamp technique to further characterize biophysical properties and physiological regulators of the Cl- current in freshly isolated guinea pig DSM cells. The Cl- current demonstrated outward rectification arising from voltage-dependent gating of Cl- channels rather than the Cl- transmembrane gradient. An exposure of DSM cells to hypotonic extracellular solution (Δ 165 mOsm challenge) did not increase the Cl- current providing strong evidence that volume-regulated anion channels do not contribute to the Cl- current in DSM cells. The Cl- current was monotonically dependent on extracellular pH, larger and lower in magnitude at acidic (5.0) and basic pH (8.5) values, respectively. Additionally, intracellularly applied phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] analog [PI(4,5)P2-diC8] increased the average Cl- current density by approximately threefold in a voltage-independent manner. The magnitude of the DSM whole cell Cl- current did not depend on the cell surface area (cell capacitance) regardless of the presence or absence of PI(4,5)P2-diC8, an intriguing finding that underscores the complex nature of Cl- channel expression and function in DSM cells. Removal of both extracellular Ca2+ and Mg2+ did not affect the DSM whole cell Cl- current, whereas Gd3+ (1 mM) potentiated the current. Collectively, our recent and present findings strongly suggest that Cl- channels are critical regulators of DSM excitability and are regulated by extracellular pH, Gd3+, and PI(4,5)P2.

Attenuated BK channel function promotes overactive bladder in a rat model of obesity.

Overactive bladder (OAB) is mostly observed in obese individuals, and is associated with enhanced excitability and contractility of the detrusor smooth muscle (DSM). Large-conductance voltage- and Ca2+-activated K+ (BK) channels reduce the excitability and contractility of the DSM. We tested whether obesity-induced OAB is associated with altered BK channel expression and activity in the DSM. Seven-week-old female Sprague-Dawley rats (N=80) were fed a normal or high-fat diet (HFD) for 12 weeks. HFD-fed rats exhibited a higher average bodyweight and urodynamically established detrusor overactivity. mRNA levels of the Kcnma1 (BKα subunit) and Kcnmb1 (BKβ1 subunit) in whole tissues and cells from the DSM were reduced in HFD-fed rats. A selective BK channel opener, NS1619, was then applied to DSM cells from the two groups of rats. Patch-clamp techniques revealed that spontaneous transient outward currents, NS1619-induced activation of spontaneous transient outward currents, and whole-cell BK currents, as well as NS1619-induced membrane hyperpolarization, were attenuated in DSM cells from HFD-fed rats. The relaxation effect of NS1619 on contractility was reduced in DSM strips from HFD-fed rats. Thus, impaired expression of Kcnma1 and Kcnmb1 in the DSM contributes to obesity-induced OAB, suggesting that BK channels could be a useful treatment targets in OAB.

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.

Properties of SK3 channel-expressing PDGFRα (+) cells in the rodent urinary bladder

Localisation of platelet-derived growth factor receptor-α (PDGFRα) (+) cells expressing small-conductance Ca2+-activated K+ (SK3) channels in the urinary bladder was investigated, while putative roles of SK3 (+) PDGFRα (+) cells in suppressing detrusor smooth muscle (DSM) spontaneous activity were explored. In guinea-pig bladder, immunohistochemistry for SK3 channels, PDGFRα or vimentin was examined, as were the effects of purinergic agonists on spontaneous phasic contractions (SPCs). In bladder of PDGFRα-GFP mice, the effects of purinergic agonists on intracellular Ca2+ signaling in PDGFRα (+) cells or DSM cells in situ and SPCs were investigated. SK3 (+) cells co-expressing PDGFRα or vimentin were distributed in DSM bundles but not inter-bundle spaces or lamina propria. SK3 (+) cells had a stellate- or spindle-shape cell body extending processes. MRS2365 (100 nM or 1 μM), a P2Y1 agonist, caused a transient contraction without inhibiting SPCs in both DSM and lamina propria. In PDGFRα-GFP mice bladder, MRS2365, (100 nM), ADP (100 μM) or ATP (100 μM) increased the Ca2+ level of PDGFRα (+) cells without suppressing spontaneous Ca2+ transients in neighboring DSM cells, and also failed to suppress SPCs. Preferential localisation of SK3 positive PDGFRα (+) cells in DSM bundles appears to indicate their functional interaction with DSM cells. However, increases in Ca2+ level of PDGFRα (+) cells upon purinergic stimulation are not associated with the inhibition of Ca2+ or contractile activity in DSM cells. Thus, it is unlikely that the SK3-dependent hyperpolarisation generated in SK3 expressing PDGFRα (+) cells is transmitted to DSMs to suppress their excitability.

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.

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.

Administration of human umbilical cord blood mononuclear cells restores bladder dysfunction in streptozotocin-induced diabetic rats.

This study evaluated the effect of human umbilical cord blood mononuclear cells (HUCB-MNCs) on bladder dysfunction in streptozotocin (STZ; 35 mg/kg, i.v.)-induced diabetic rats. Adult male Sprague-Dawley rats (n = 30) were equally divided into three groups: control group, STZ-diabetic group, and HUCB-MNC-treated group (1 × 106 cells). HUCB-MNCs were isolated by density gradient centrifugation from eight healthy donors and injected into the corpus cavenosum in STZ-diabetic rats 4 weeks after the induction of diabetes. Studies were performed 4 weeks after HUCB-MNC or vehicle injection. In vitro organ bath studies were performed on bladder strips, whereas protein expression of hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), and α-smooth muscle actin (SMA) in the bladder and the ratio of smooth muscle cells (SMCs) to collagen were determined using western blotting and Masson trichrome staining. Neurogenic contractions of detrusor smooth muscle strips were 55% smaller in the diabetic group than control group (P < 0.05); these contractions were normalized by HUCB-MNC treatment. In addition, HUCB-MNC treatment restored the impaired maximal carbachol-induced contractile response in detrusor strips in the diabetic group (29%; P < 0.05). HUCB-MNC treatment improved the KCl-induced contractile response in the diabetic bladder (68%; P < 0.05), but had no effect on ATP-induced contractile responses. Increased expression of HIF-1α and VEGF protein and decreased expression of α-SMA protein and the SMC/collagen ratio in diabetic rats were reversed by HUCB-MNC. Administration of HUCB-MNCs facilitates bladder function recovery, which is likely related to downregulation of HIF-1α expression and attenuation of fibrosis in STZ-diabetic rats.

Influence of sildenafil on the purinergic components of nerve-mediated and urothelial ATP release from the bladder of normal and spinal cord injured mice.

Background and purposePDE inhibitors such as sildenafil alleviate lower urinary tract symptoms; however, a complete understanding of their action on the bladder remains unclear. We are investigating the effects of sildenafil, on post and preganglionic nerve‐mediated contractions of the mouse bladder, and neuronal and urothelial ATP release.Experimental approachBladders were used from young (12 weeks), aged (24 months), and spinal cord transected (SCT), mice, for in vitro contractility experiments. An arterially perfused in situ whole mouse model was used to record bladder pressure. Nerve‐mediated contractions were generated by electrical field stimulation (EFS) of postganglionic nerve terminals or the pelvic nerve. ATP release during EFS in intact detrusor strips, and during stretch of isolated mucosa strips, was measured using a luciferin‐luciferase assay.Key resultsSildenafil (20 μM) inhibited nerve‐mediated contractions in young mice, with an increase in f 1/2 values in force–frequency relationships, demonstrating a greater effect at low frequencies. Sildenafil reduced the atropine‐resistant, purinergic component of nerve‐mediated contractions, and suppressed neuronal ATP release upon EFS in vitro. Sildenafil reduced the preganglionic pelvic nerve stimulated bladder pressure recordings in situ; comparable to in vitro experiments. Sildenafil reduced stretch‐induced urothelial ATP release. Sildenafil also relaxed nerve‐mediated contractions in aged and SCT mice.Conclusion and implicationsSildenafil has a greater effect on the low‐frequency, purinergic‐mediated contractions and suppresses neuronal ATP release. In addition, sildenafil reduces stretch‐induced urothelial ATP release. These results demonstrate a novel action of sildenafil to selectively inhibit ATP release from nerve terminals innervating detrusor smooth muscle and the urothelium.

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.

β3 -Adrenoceptors in the normal and diseased urinary bladder-What are the open questions?

β3 -Adrenoceptor agonists are used in the treatment of overactive bladder syndrome. Although the relaxant response to adrenergic stimulation in human detrusor smooth muscle cells is mediated mainly via β3 -adrenoceptors, the plasma concentrations of the therapeutic dose of mirabegron, the only clinically approved β3 -adrenoceptor agonist, are considerably lower than the EC50 for causing direct relaxation of human detrusor, suggesting a mechanism of action other than direct relaxation of detrusor smooth muscle. However, the site and mechanism of action of β3 -adrenoceptor agonists in the bladder have not been firmly established. Postulated mechanisms include prejunctional suppression of ACh release from the parasympathetic nerves during the storage phase and inhibition of micro-contractions through β3 -adrenoceptors on detrusor smooth muscle cells or suburothelial interstitial cells. Implications of possible desensitization of β3 -adrenoceptors in the bladder upon prolonged agonist exposure and possible causes of rarely observed cardiovascular effects of mirabegron are also discussed. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.

A computational model of large conductance voltage and calcium activated potassium channels: implications for calcium dynamics and electrophysiology in detrusor smooth muscle cells.

The large conductance voltage and calcium activated potassium (BK) channels play a crucial role in regulating the excitability of detrusor smooth muscle, which lines the wall of the urinary bladder. These channels have been widely characterized in terms of their molecular structure, pharmacology and electrophysiology. They control the repolarising and hyperpolarising phases of the action potential, thereby regulating the firing frequency and contraction profiles of the smooth muscle. Several groups have reported varied profiles of BK currents and I-V curves under similar experimental conditions. However, no single computational model has been able to reconcile these apparent discrepancies. In view of the channels' physiological importance, it is imperative to understand their mechanistic underpinnings so that a realistic model can be created. This paper presents a computational model of the BK channel, based on the Hodgkin-Huxley formalism, constructed by utilising three activation processes - membrane potential, calcium inflow from voltage-gated calcium channels on the membrane and calcium released from the ryanodine receptors present on the sarcoplasmic reticulum. In our model, we attribute the discrepant profiles to the underlying cytosolic calcium received by the channel during its activation. The model enables us to make heuristic predictions regarding the nature of the sub-membrane calcium dynamics underlying the BK channel's activation. We have employed the model to reproduce various physiological characteristics of the channel and found the simulated responses to be in accordance with the experimental findings. Additionally, we have used the model to investigate the role of this channel in electrophysiological signals, such as the action potential and spontaneous transient hyperpolarisations. Furthermore, the clinical effects of BK channel openers, mallotoxin and NS19504, were simulated for the detrusor smooth muscle cells. Our findings support the proposed application of these drugs for amelioration of the condition of overactive bladder. We thus propose a physiologically realistic BK channel model which can be integrated with other biophysical mechanisms such as ion channels, pumps and exchangers to further elucidate its micro-domain interaction with the intracellular calcium environment.

The effect of endoplasmic reticulum stress on detrusor muscle apoptosis in diabetic cystopathy

Objective To observe the effect of Endoplasmic Reticulum Stress (ERS) on detrusor muscle apoptosis in Streptozotocin (STZ)-induced diabetic rats. Methods At 4, 8, 12, and 16 weeks after induction of diabetic rat models, the sections of detrusor muscle tissue were made and were stained with Van Gieson solution, the collagen fibers of each group were observed and their proportions were calculated; the sub-cellular ultrastructure was observed by Transmission Electron Microscopy (TEM). Proliferating Cell Nuclear Antigen (PCNA) assay kit and Strept Avidin-Biotin Complex (SABC) staining were used for the evaluation of Detrusor Smooth Muscle (DSM) cell proliferation. Moreover, the expression of three hallmarks of ERS-associated apoptosis, including Glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and cysteinyl aspartate-specific protease (Caspase)-12, was detected by Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Results The proportion of collagen fibers showed a significant climbing trend [(13.38±2.65)%, (18.14±1.93)%, (19.71±3.05)% vs. (7.86±0.72)%, P<0.05] since DM8w group, except DM4w group [(8.02±0.43)%]. With the time prolonged, the swelled and fused cisternaes started to show; the mild particle fusion and degranulation also could be seen in the zone of ER; the distorted or pyknotic nucleus contained clusters of unidentified dark-stained granules that could be observed. Compared with the control group, the increased PCNA-positive percentages [(20.34±4.55)%, (46.88±7.12)%, (39.78±1.93)%, (22.15±5.64)% vs. (16.26±2.43)%, P<0.05] and apoptosis indexes [(17.12±2.78)%, (28.12±4.09)%, (28.12±4.09)%, (43.39±6.82)% vs. (6.32±1.23)%, P<0.05] can be seen in model groups. Elevated expression of GRP78, CHOP, and Caspase-12 at both protein and mRNA levels in a time-dependent fashion were detected (P<0.05). Conclusion Morphological and ultrastructural damages to the DSM as well as the cell proliferation and apoptosis indicate the time-dependent alterations. The occurrence of ERS may be involved in the development of diabetic cystopathy (DCP) and may contribute to the diabetic detrusor muscle apoptosis. Key words: Endoplasmic reticulum stress; Diabetic cystopathy; Detrusor; Apoptosis

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 PDE9 inhibitor BAY 73‐6691 in the contractile response of cavernosal and detrusor smooth muscle of sickle cell disease mice

IntroductionGenitourinary tract function is affected in sickle cell disease (SCD). Modulation of cyclic guanosine monophosphate (cGMP)‐dependent signalling is important for the contractile mechanism of detrusor (DSM) and corpus cavernosum (CC) smooth muscle and may characterize a therapeutic target for theses manifestations in the SCD. Phosphodiesterase‐9 (PDE9) specifically hydrolyzes cGMP, and was detected in CC and bladder urothelium. BAY 73‐6691, selective PDE9 inhibitor, has been used for understanding the role of cGMP signaling in the SCD. However, no previous studies have explored the selective PDE9 inhibition with BAY 73‐6691 in the DSM and CC in SCD.ObjectiveThe aim of this study was to investigate the effect of BAY 73‐6691 on the contractile response of CC and DSM of SCD mice.MethodsBerkeley transgenic sickle cell mice (SS) and C57BL/6 mice (control) were used. Cumulative concentration‐response curves to muscarinic receptor agonist carbachol (CCh; 0.01–100 μM), chloride potassium (KCl; 1–300 mM) and frequency‐response curves for electrical field stimulation (EFS; 1–32 Hz) were obtained in strips of DSM from both control and SS mice. In the CC, cumulative concentration‐response curves to α1‐adrenoceptor agonist phenylephrine (PE; 0.01–100 μM) and frequency‐response curves for electrical field stimulation (EFS; 1–32 Hz) were carried out in the absence and presence of BAY 73‐6691 (100 nM and 1 μM; 30 min).ResultsIn the evaluation of DSM, the SS mice presented an increased in the contractile response mediated by CCh (P&lt;0.05), when compared to control mice. Surprisingly, BAY 73‐6691 increased the contraction mediated by CCh in DSM of SS mice (p&lt;0.05), but did not alter the contraction response in the control mice. Contractile responses induced by KCl were increased in the SS mice (p&lt;0.001) compared to control mice. However, no significant differences for BAY 73‐6671 incubation were found between control and SS group. EFS‐induced neurogenic contractions in the DSM of SS mice were 90% higher in the frequency of 32 Hz (p&lt;0.01) compared to control. Pre‐incubation with BAY 73‐6671 no affect the neurogenic contraction in the control and SCD group. Cavernosal contractile to PE were higher in SS mice compared to control mice (P&lt;0.01). Similarly to DSM, BAY 73‐6691 increased the contraction mediated by PE in CC of SS mice (p&lt;0.05), but did not alter the contraction response in control mice. However, EFS‐induced neurogenic contractions in CC of SS mice were 49% lower in the frequency of 32 Hz (p&lt;0.01) compared to the control. BAY 73‐6691 did not change the neurogenic contraction in both groups.ConclusionOur preliminary data shows that, BAY 73‐6691 promotes increase of contractile response in SS mice, acting in the muscarinic and adrenergic signaling pathway in the DSM and CC, respectively. Further experiments are needed to identify the exact mechanisms of drug‐induced alterations in the contraction of the DSM and CC in SCD.Support or Funding InformationFAPESPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MP11-15 CHRONIC HIGH FAT DIET IMPAIRS DETRUSOR MITOCHONDRIAL FATTY ACID OXIDATION IN MALE BUT NOT FEMALE MICE

INTRODUCTION AND OBJECTIVES:Obesity increases the prevalence of bladder dysfunction in both males and females. The bladder’s mucosal and detrusor smooth muscle layers are involved in bladder signaling and contraction, respectively. The underlying causes of obesity-induced bladder dysfunction in males and females are poorly understood. Mitochondrial dysfunction is common in the heart, liver and skeletal muscle with high fat diet. This study aims to determine the impact of long-term high fat diet (HFD) on bladder mucosal and detrusor mitochondrial respiration in male and female mice.METHODS:Adult male and female mice were fed a control (10% kcal fat) or HFD (45% kcal fat; n=7/group) for 24 weeks. Bladders were collected, weighed and separated into mucosal and detrusor layers. Tissues were cut into strips, permeabilized, and placed in an OROBOROS Oxygraphy-2K to measure respiration. Substrates (pyruvate/malate, glutamate, L-octanoylcarnitine/malate, or succinate/rotenone) were added with increasing concentra...

Chronic high fat diet lowers male detrusor mitochondrial fatty acid oxidation while females are protected

IntroductionObesity increases the prevalence of bladder dysfunction in both males and females. The bladder's mucosal and detrusor smooth muscle layers are involved in bladder signaling and contraction, respectively. The underlying causes of obesity‐induced bladder dysfunction in males and females are poorly understood. Mitochondrial dysfunction is common in the heart, liver and skeletal muscle with high fat diet (HFD). This study will characterize sex differences in mitochondrial respiration in bladder mucosal and detrusor layers. Additionally, we will determine the impact of long‐term HFD on bladder mitochondrial respiration in male and female mice.MethodsMale and female mice (n=7/group, 20–24 wks) were used to assess bladder mitochondrial sex differences. A second group of male and female mice (10 wks, n=5–7/group) were fed a control (10% kcal fat) or HFD (45% kcal fat) for 24 weeks. Bladders were collected, weighed and separated into mucosal and detrusor layers. Tissues were cut into strips, permeabilized, and placed in an OROBOROS Oxygraphy‐2K to measure respiration using a modified version of the creatine kinase energetic clamp. Substrates (pyruvate/malate, glutamate, L‐octanoylcarnitine/malate, or succinate/rotenone) were added followed by increasing concentrations of phosphocreatine to allow for substrate‐specific bioenergetic analyses across a physiological span of ATP free energies. Respiratory conductance was calculated for each substrate based on the rate of change in respiration for a given increase in ATP free energy.ResultsBladder respiration and respiratory conductance in the presence of L‐octanoylcarnitine/malate in mucosal and detrusor tissue was significantly decreased in males (p&lt;0.05). Respiration and respiratory conductance remained unchanged between sexes in the presence of pyruvate/malate, glutamate, and succinate/rotenone. Chronic HFD increased body weight and decreased the bladder to body weight ratio in both sexes (p&lt;0.05). In HFD males, mucosal respiration and respiratory conductance were decreased in the presence of L‐octanoylcarnitine/malate (p&lt;0.05). In contrast, pyruvate/malate, glutamate, and succinate/rotenone did not change HFD male respiration and respiratory conductance. In HFD females, respiration and respiratory conductance were unchanged in presence of pyruvate/malate, glutamate, and L‐octanoylcarnitine/malate. However, HFD female mucosal layer had decreased respiration with succinate/rotenone but no change in respiratory conductance.ConclusionsMale mice have lower baseline fatty acid metabolism within the bladder's detrusor smooth muscle layer compared to females. With chronic HFD, male fatty acid metabolism within the mucosal and detrusor muscle layers is further lowered whereas female mice are protected. Ongoing studies will determine if excess lipids accumulate within the bladder and if we can rescue fatty acid metabolism with co‐factors such as L‐carnitine or acetyl‐CoA. Understanding bladder metabolic and bioenergetic sex differences will help to create more effective treatments for bladder dysfunctionSupport or Funding InformationNIDDK DiaComp, ECU SBRP, APS UGSRFThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.