Detrusor Smooth Muscle Research Articles

The Association between Specimen Neuromuscular Characteristics and Urinary Incontinence after Robotic-Assisted Radical Prostatectomy.

RARP specimens from 29 men who underwent bilateral nerve sparing were reanalyzed. Urinary incontinence was evaluated using the International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF) at 6 weeks post surgery and last follow-up. Linear and logistic regression analyses were performed to assess neuromuscular characteristics and incontinence. At the 1-year follow-up, 11 patients (38%) reported severe incontinence (>12 ICIQ-SF score). The median number of peripheral nerves observed at the base and apex in the specimens was 52 (IQR 13-139) and 59 (IQR: 28-129), respectively. Ganglia were present in 19 patients (65%) at the base and 12 patients (41%) at the apex. Additionally, the median proportional area of detrusor smooth muscle fibers at the base was 0.54 (IQR 0.31-1), while the median proportional area of striated muscle fibers at the apex was 0.13 (IQR 0.08-0.24). No statistically significant associations were found. Histologic neuromuscular characteristics were not associated with postoperative urinary incontinence. Enhanced intraoperative evaluation and larger-scale studies may prove useful for the prediction of postprostatectomy incontinence.

Exploring the Genetic Risk of Childhood Daytime Urinary Incontinence: A Genome-Wide Association Study.

Childhood incontinence is stigmatized and underprioritized, and a basic understanding of its pathogenesis is missing. Our goal was to identify risk-conferring genetic variants in daytime urinary incontinence (DUI). We conducted a genome-wide association study in the Danish iPSYCH2015 cohort. Cases (3024) were identified through DUI diagnosis codes and redeemed prescriptions for DUI medication in children aged 5 to 20. Controls (30,240), selected from the same sample, were matched to cases on sex and psychiatric diagnoses, if any, and down-sampled to a 1:10 case:control ratio. Replication was performed in the Icelandic deCODE cohort (5475 cases/287,773 controls). Single-nucleotide polymorphism heritability was calculated using the genome-based restricted maximum likelihood method. Cross-trait genetic correlation was estimated using linkage disequilibrium score regression. Polygenic risk scores generated with LDpred2-auto and BOLT-LMM were assessed for association. Variants on chromosome 6 (rs12210989, OR = 1.24 [95% CI: 1.17-1.32], P = 3.21 × 10-12) and 20 (rs4809801, OR = 1.18 [95% CI: 1.11-1.25], P = 3.66 × 10-8) reached genome-wide significance and implicated the PRDM13 and RIPOR3 genes. Chromosome 6 findings were replicated (P = .024, OR = 1.09 [95% CI: 1.01-1.16]). Liability scale heritability ranged from 10.20% (95% CI: 6.40%-14.00%) to 15.30% (95% CI: 9.66%-20.94%). DUI and nocturnal enuresis showed positive genetic correlation (rg = 1.28 ± 0.38, P = .0007). DUI was associated with attention-deficit/hyperactivity disorder (OR = 1.098 [95% CI: 1.046-1.152], P < .0001) and body mass index (OR = 1.129 [95% CI: 1.081-1.178], P < .0001) polygenic risk. Common genetic variants contribute to the risk of childhood DUI and genes important in neuronal development and detrusor smooth muscle activity were implicated. These findings may help guide identification of new treatment targets.

Contribution of transient receptor potential vanilloid 1 (TRPV1) channel to cholinergic contraction of rat bladder smooth muscle.

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-permeable ion channel that is gated by the pungent constituent of red chili pepper, capsaicin, and by related chemicals from the group of vanilloids, in addition to noxious heat. It is expressed mostly in sensory neurons to act as a detector of painful stimuli produced by pungent chemicals and high temperatures. Although TRPV1 is also found outside the sensory nervous system, its expression and function in the bladder detrusor smooth muscle (DSM) remain controversial. Here, by using Ca2+ imaging and patch clamp on isolated rat DSM cells, in addition to tensiometry on multicellular DSM strips, we show that TRPV1 is expressed functionally in only a fraction of DSM cells, in which it acts as an endoplasmic reticulum Ca2+-release channel responsible for the capsaicin-activated [Ca2+]i rise. Carbachol-stimulated contractions of multicellular DSM strips contain a TRPV1-dependent component, which is negligible in the circular DSM but reaches ≤50% in the longitudinal DSM. Activation of TRPV1 in rat DSM during muscarinic cholinergic stimulation is ensured by phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists. Immunofluorescence detection of TRPV1 protein in bladder sections and isolated DSM cells confirmed both its preferential expression in the longitudinal DSM sublayer and its targeting to the endoplasmic reticulum. We conclude that TRPV1 is an essential contributor to the cholinergic contraction of bladder longitudinal DSM, which might be important for producing spatial and/or temporal anisotropy of bladder wall deformation in different regions during parasympathetic stimulation. KEY POINTS: The transient receptor potential vanilloid 1 (TRPV1) heat/capsaicin receptor/channel is localized in the endoplasmic reticulum membrane of detrusor smooth muscle (DSM) cells of the rat bladder, operating as a calcium-release channel. Isolated DSM cells are separated into two nearly equal groups, within which the cells either show or do not show TRPV1-dependent [Ca2+]i rise. Carbachol-stimulated, muscarinic ACh receptor-mediated contractions of multicellular DSM strips contain a TRPV1-dependent component. This component is negligible in the circular DSM but reaches ≤50% in longitudinal DSM. Activation of TRPV1 in rat DSM during cholinergic stimulation involves phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists.

Cellular and Molecular Aspect of Bladder Pain Syndrome: An Entry Point to Exploration of Its Pathogenesis

&amp;lt;i&amp;gt;Background:&amp;lt;/i&amp;gt; Bladder pain syndrome/interstitial cystitis (BPS/IC) can cause pelvic pain, frequent urination, and a strong urge to urinate. These symptoms can significantly reduce quality of life, causing psychological distress, sexual dysfunction, poor sleep quality, decreased work productivity, and increased morbidity. Despite the prevalence of this condition, determining the most effective treatment guidelines for BPS/IC remains a challenge due to the complexity of its pathogenesis. &amp;lt;i&amp;gt;Objective:&amp;lt;/i&amp;gt; Understanding cellular and molecular aspects is essential to explore different cell types in changes in function and sensitivity of the urothelial layer and chronic inflammation. &amp;lt;i&amp;gt;Main Ideas:&amp;lt;/i&amp;gt; Cellular aspects in the pathogenesis of BPS/IC include Umbrella Cells, Basal and Intermediate Cells, Paraneuron Cells, Myofibroblasts and Telocytes, Detrusor Smooth Muscle Cells, Nerve Cells, Astrocytes, Microglia, CD68+ Macrophages, CD74+ Lymphocytes, Eosinophils, and Mast Cells. Disruption of these cells leads to altered urothelial barrier function, sensitivity, and chronic inflammation. Molecular aspects include chronic inflammation with increases in p38-mitogen activated protein kinase (p38 MAPK), Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α), Nerve Growth Factor (NGF), Brain-Derived Neurothropic Peptide (BDNF), and other molecules. &amp;lt;i&amp;gt;Conclusion:&amp;lt;/i&amp;gt; Changes in the urothelial barrier and bladder wall sensitivity are also significant. Complex interactions between the immune and nervous systems contribute to chronic inflammation through positive feedback. Therefore, this article aims to understand the cellular and molecular aspects that play a role in the pathogenesis of BPS/IC and help provide appropriate treatment.

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.

Immunomodulatory imide drugs inhibit human detrusor smooth muscle contraction and growth of human detrusor smooth muscle cells, and exhibit vaso-regulatory functions

BackgroundThe immunomodulatory imide drugs (IMiDs) thalidomide, lenalidomide and pomalidomide may exhibit therapeutic efficacy in the prostate. In lower urinary tract symptoms (LUTS), voiding and storage disorders may arise from benign prostate hyperplasia, or overactive bladder. While current therapeutic options target smooth muscle contraction or cell proliferation, side effects are mostly cardiovascular. Therefore, we investigated effects of IMiDs on human detrusor and porcine artery smooth muscle contraction, and growth-related functions in detrusor smooth muscle cells (HBdSMC). MethodsCell viability was assessed by CCK8, and apoptosis and cell death by flow cytometry in cultured HBdSMC. Contractions of human detrusor tissues and porcine interlobar and coronary arteries were induced by contractile agonists, or electric field stimulation (EFS) in the presence or absence of an IMID using an organ bath. Proliferation was assessed by EdU assay and colony formation, cytoskeletal organization by phalloidin staining, ResultsDepending on tissue type, IMiDs inhibited cholinergic contractions with varying degree, up to 50 %, while non-cholinergic contractions were inhibited up to 80 % and 60 % for U46619 and endothelin-1, respectively, and EFS-induced contractions up to 75 %. IMiDs reduced viable HBdSM cells in a time-dependent manner. Correspondingly, proliferation was reduced, without showing pro-apoptotic effects. In parallel, IMiDs induced cytoskeletal disorganization. ConclusionsIMiDs exhibit regulatory functions in various smooth muscle-rich tissues, and of cell proliferation in the lower urinary tract. This points to a novel drug class effect for IMiDs, in which the molecular mechanisms of action of IMiDs merit further consideration for the application in LUTS.

In Silico Electrophysiological Investigation of Transient Receptor Potential Melastatin-4 Ion Channel Biophysics to Study Detrusor Overactivity.

Enhanced electrical activity in detrusor smooth muscle (DSM) cells is a key factor in detrusor overactivity which causes overactive bladder pathological disorders. Transient receptor potential melastatin-4 (TRPM4) channels, which are calcium-activated cation channels, play a role in regulating DSM electrical activities. These channels likely contribute to depolarizing the DSM cell membrane, leading to bladder overactivity. Our research focuses on understanding TRPM4 channel function in the DSM cells of mice, using computational modeling. We aimed to create a detailed computational model of the TRPM4 channel based on existing electrophysiological data. We employed a modified Hodgkin-Huxley model with an incorporated TRP-like current to simulate action potential firing in response to current and synaptic stimulus inputs. Validation against experimental data showed close agreement with our simulations. Our model is the first to analyze the TRPM4 channel's role in DSM electrical activity, potentially revealing insights into bladder overactivity. In conclusion, TRPM4 channels are pivotal in regulating human DSM function, and TRPM4 channel inhibitors could be promising targets for treating overactive bladder.

MRI-based radiomic features of the urinary bladder wall identify patients with moderate-to-severe international prostate symptom score

BackgroundThe International Prostate Symptom Score (IPSS) is a patient-reported measurement to assess the lower urinary tract symptoms of bladder outlet obstruction. Bladder outlet obstruction induces molecular and morphological alterations in the urothelium, suburothelium, detrusor smooth muscle cells, detrusor extracellular matrix, and nerves. We sought to analyze MRI-based radiomics features of the urinary bladder wall and their association with IPSS.MethodIn this retrospective study, 87 patients who had pelvic MRI scans were identified. A biomarker discovery approach based on the optimal biomarker (OBM) method was used to extract features of the bladder wall from MR images, including morphological, intensity-based, and texture-based features, along with clinical variables. Mathematical models were created using subsets of features and evaluated based on their ability to discriminate between low and moderate-to-severe IPSS (less than 8 vs. equal to or greater than 8).ResultsOf the 7,666 features per patient, four highest-ranking optimal features were derived (all texture-based features), which provided a classification accuracy of 0.80 with a sensitivity, specificity, and area under the receiver operating characteristic curve of 0.81, 0.81, and 0.87, respectively.ConclusionA highly independent set of urinary bladder wall features derived from MRI scans were able to discriminate between patients with low vs. moderate-to-severe IPSS with accuracy of 80%. Such differences in MRI-based properties of the bladder wall in patients with varying IPSS’s might reflect differences in underlying molecular and morphological alterations that occur in the setting of chronic bladder outlet obstruction.

Can Urinary Bladder Innervation Be Restored After Outlet Obstruction and Denervation?

Transurethral resection of the prostate, or other methods to decrease outlet resistance usually leads to relief of symptoms in patients with bladder outlet obstruction (BOO). If symptoms of underactivity persist after normalization of outflow conditions, treatment options are limited. In this review, we hypothesize, based on results from basic research, what might become treatment options for such patients in the future. The primary local treatment will still aim at reducing outlet obstruction. We speculate that local secondary treatment in the future might include transplantation of stem cells or mature bladder ganglion cells into the bladder wall. There has been some success in transplanting ganglion cells into the rat bladder. The ganglion cells will sprout into the surrounding tissue but functional connections between the axons of the transplanted neurons, and the detrusor smooth muscle have so far not been demonstrated. Neurotrophins or neurotrimin might be injected into the bladder wall to increase the sprouting of existing or transplanted neurons. Stem cell transplantation has been performed and improves detrusor function, but it has so far, been difficult to demonstrate transplanted stem cells. BOO, persisting detrusor underactivity, and decreased nerve density are often combined with inflammatory activity of the lower urinary tract. NLR family pyrin domain containing 3 (NLRP3) and its messenger RNA (mRNA) as well as cyclooxygenase-2 (Cox-2) mRNA are increased in obstructed bladders. Systemic treatment with the NLRP3 inhibitor glyburide normalized nerve density in rat bladder, and, to some extent, bladder function. It is unclear whether Cox-2 is involved in the decreased nerve density following obstruction, but Cox-2 mRNA increases 5-fold in obstructed bladder. Future therapy against bladder underactivity remaining following relief of obstruction includes either systemic treatment, perhaps by anti-inflammatory drugs, or local treatment by injection of stem cells, mature ganglion cells, and/or neurotrophins or neurotrimin into the bladder wall.

Piezo1-dependent inhibition of background potassium current in the rat bladder smooth muscle cells

The smooth muscle layer of the urinal bladder (detrusor), along with the urothelium, has autonomous mechanosensitivity and serves as a main sensitive receptor in the organ. Piezo1, together with TREK-1 channel, could play a role of local mechanoreceptors in bladder detrusor smooth muscle (DSM) cells. Piezo1 is a so-called “true” mechanosensitive calcium-permeable ion channel, sensitive to pressure, shear stress, and is activated by pharmacological agonist Yoda1. Using patch-clamp and microfluorescence calcimetry, an unexpected effect from Yoda1 was shown, that is inhibition of rest K+ currents at depolarizing command voltage up to +80 mV. The functional presence of Piezo1 is confirmed by Yoda1-induced rise of intracellular Ca2+ concentration in DSM cells, this was visualized using a Ca2+ sensitive dye Fluo-4 AM, and polymerase cyclic reaction with reverse transcription. In conclusion, Piezo1 channels present in DSM cells and are selectively activated with Yoda1, that causes the inhibition of resting potassium currents.

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

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

Hydrogen Sulfide (H2S) and Reactive Oxygen Species (ROS) Scavengers Have a Protective Effect on Carbachol-induced Contractions That are Impaired by High Glucose in Detrusor Smooth Muscle

Hydrogen Sulfide (H2S) and Reactive Oxygen Species (ROS) Scavengers Have a Protective Effect on Carbachol-induced Contractions That are Impaired by High Glucose in Detrusor Smooth Muscle

Роль К+-провідного каналу TREK-1 у механочутливості гладеньком’язових клітин детрузора сечового міхура щура

Currently, TREK-1 is considered to be the main mechanosensitive channel in detrusor smooth muscle (DSM) cells. The aim of our study was to detect the functioning of the K+-conducting mechanosensitive TREK-1 channel in rat DSM cells using the patch-clamp technique in response to hydrodynamic stimulation (shear stress) and to determine the effects of a TREK-1 agonist – arachidonic acid (AA) and an antagonist – L-methionine. Mechanical stimulation of DSM cells using hydrodynamic stress led to the appearance of a membrane current with signs of pronounced outward rectification at positive membrane potentials, which is typical of TREK-1 activation. The application of AA (50 mcmol/l) activated a current with similar characteristics of the outward rectification to the shear stress-activated one. L-methionine (10 mcmol/l) almost completely prevented the generation of an outwardly rectifying current in response to shear stress stimulation. DSM cells also retained the ability to generate a mechanoactivated current with a more pronounced inward component when extracellular and intracellular K+ were replaced by Cs+. It was concluded that the dominant mechanoactivated current in rat DSM cells is carried by K+-selective TREK-1 channels, but a small portion of this current can also be carried by other nonselective mechanosensitive cation channels.

MP09-11 Marked Increase in Mitochondrial Respiration, Hydrogen Peroxide Emitting Potential and ATP Production in the Urothelium Versus Detrusor Smooth Muscle.

MP09-11 Marked Increase in Mitochondrial Respiration, Hydrogen Peroxide Emitting Potential and ATP Production in the Urothelium Versus Detrusor Smooth Muscle.

Impact of intravascular hemolysis on functional and molecular alterations in the urinary bladder: implications for an overactive bladder in sickle cell disease.

Patients with sickle cell disease (SCD) display an overactive bladder (OAB). Intravascular hemolysis in SCD is associated with various severe SCD complications. However, no experimental studies have evaluated the effect of intravascular hemolysis on bladder function. This study aimed to assess the effects of intravascular hemolysis on the micturition process and the contractile mechanisms of the detrusor smooth muscle (DSM) in a mouse model with phenylhydrazine (PHZ)-induced hemolysis; furthermore, it aimed to investigate the role of intravascular hemolysis in the dysfunction of nitric oxide (NO) signaling and in increasing oxidative stress in the bladder. Mice underwent a void spot assay, and DSM contractions were evaluated in organ baths. The PHZ group exhibited increased urinary frequency and increased void volumes. DSM contractile responses to carbachol, KCl, α-β-methylene-ATP, and EFS were increased in the PHZ group. Protein expression of phosphorylated endothelial NO synthase (eNOS) (Ser-1177), phosphorylated neuronal NO synthase (nNOS) (Ser-1417), and phosphorylated vasodilator-stimulated phosphoprotein (VASP) (Ser-239) decreased in the bladder of the PHZ group. Protein expression of oxidative stress markers, NOX-2, 3-NT, and 4-HNE, increased in the bladder of the PHZ group. Our study shows that intravascular hemolysis promotes voiding dysfunction correlated with alterations in the NO signaling pathway in the bladder, as evidenced by reduced levels of p-eNOS (Ser-1177), nNOS (Ser-1417), and p-VASP (Ser-239). The study also showed that intravascular hemolysis increases oxidative stress in the bladder. Our study indicates that intravascular hemolysis promotes an OAB phenotype similar to those observed in patients and mice with SCD.

Современные возможности лечения доброкачественной гиперплазии предстательной железы и симптомов нижних мочевыводящих путей: новые данные и опыт клинического применения гексанового экстракта плодов Serenoa repens

The article discusses the pharmacological properties and results concerning the clinical use of a medicinal herbal preparation — hexane extract of Serenoa repens (HESr) (creeping palm fruit). In recent years, quite a lot of research data has been accumulated indicating a multicomponent mechanism of drug action. It has been found to have antiandrogenic, antiproliferative and anti-inflammatory effects in the tissues of the prostate gland (pancreas). Studies have been published describing a new mechanism of action of HESr — antineurogenic, the implementation of which helps to reduce infravesical obstruction, leads to inhibition of α1-adrenergic, thromboxane-induced and cholinergic contraction of the pancreatic and detrusor smooth muscles, and also inhibits the growth of pancreatic stromal cells. At present, there is evidence proving the highest clinical efficacy level of the HESr (data from several meta-analyses). The listed effects and patterns of HESr are illustrated by clinical examples of prescribing this drug to patients with lower urinary tract symptoms and benign pancreatic hyperplasia. The safety of use in conditions of polypragmasy in a comorbid patient, the possibility of long-term use without the adverse events occurrence and ensuring a higher life quality while maintaining full sexual function are also particularly noted. KEYWORDS: lower urinary tract symptoms, benign prostatic hyperplasia, chronic bacterial prostatitis, hexane extract of Serenoa repens fruit, ejaculation, life quality. FOR CITATION: Amdiy R.E., Simanov R.N. Modern treatment options for benign prostatic hyperplasia and lower urinary tract symptoms: new data and clinical picture of the hexane extract of Serenoa repens fruit. Russian Medical Inquiry. 2024;8(4):228–234 (in Russ.). DOI: 10.32364/2587-6821-2024-8-4-7.

TRPA1 channel mediates methylglyoxal-induced mouse bladder dysfunction.

Introduction: The transient receptor potential ankyrin 1 channel (TRPA1) is expressed in urothelial cells and bladder nerve endings. Hyperglycemia in diabetic individuals induces accumulation of the highly reactive dicarbonyl compound methylglyoxal (MGO), which modulates TRPA1 activity. Long-term oral intake of MGO causes mouse bladder dysfunction. We hypothesized that TRPA1 takes part in the machinery that leads to MGO-induced bladder dysfunction. Therefore, we evaluated TRPA1 expression in the bladder and the effects of 1 h-intravesical infusion of the selective TRPA1 blocker HC-030031 (1nmol/min) on MGO-induced cystometric alterations. Methods: Five-week-old female C57BL/6 mice received 0.5% MGO in their drinking water for 12weeks, whereas control mice received tap water alone. Results: Compared to the control group, the protein levels and immunostaining for the MGO-derived hydroimidazolone isomer MG-H1 was increased in bladders of the MGO group, as observed in urothelium and detrusor smooth muscle. TRPA1 protein expression was significantly higher in bladder tissues of MGO compared to control group with TRPA1 immunostaining both lamina propria and urothelium, but not the detrusor smooth muscle. Void spot assays in conscious mice revealed an overactive bladder phenotype in MGO-treated mice characterized by increased number of voids and reduced volume per void. Filling cystometry in anaesthetized animals revealed an increased voiding frequency, reduced bladder capacity, and reduced voided volume in MGO compared to vehicle group, which were all reversed by HC-030031 infusion. Conclusion: TRPA1 activation is implicated in MGO-induced mouse overactive bladder. TRPA1 blockers may be useful to treat diabetic bladder dysfunction in individuals with high MGO levels.

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.

Long-term aspirin administration suppresses inflammation in diabetic cystopathy.

Diabetic cystopathy (DCP) is one of the most common and troublesome urologic complications of diabetes mellitus, characterized by chronic low-grade inflammatory response. However, the correlation between inflammation and disease progression remains ambiguous and effective drugs interventions remain deficient. Herein, during 12-week study, 48 male Sprague-Dawley rats were randomly assigned to four groups: negative control (NC), NC treated with aspirin (NC+Aspirin), DCP, and DCP treated with aspirin (DCP+Aspirin). Type 1 diabetes mellitus was established by intraperitoneal injection of streptozotocin (65 mg/kg). After 2 weeks modeling, the rats in treatment groups received daily oral aspirin (100 mg/kg/d). After 10 weeks of treatment, aspirin ameliorated pathological weight loss and bladder weight increase in diabetic rats, accompanied by a 16.5% decrease in blood glucose concentrations. H&E, Masson, immunohistochemistry and transmission electron microscopy revealed that a dilated bladder with thickened detrusor smooth muscle (DSM) layer, inflammatory infiltration, fibrosis and ultrastructural damage were observed in diabetic rats, which were obviously ameliorated by aspirin. The dynamic investigations at 4, 7 and 10 weeks revealed inflammation gradually increased as the disease progresses. After 10 weeks of treatment, the expression of TNF-α, IL-1β, IL-6, and NF-κB has been decreased to 78%, 39.7%, 44.1%, 33.3% at mRNA level and 67.6%, 76.7%, 71.4%, 67.1% at protein level, respectively (DCP+Aspirin vs. DCP, p < 0.01). Aspirin partially restored the increased expression of inflammatory mediators in bladder DSM of diabetic rats. The study provided insight into long-term medication therapies, indicating that aspirin might serve as a potential strategy for DCP treatment.