Urinary Bladder Smooth Muscle Research Articles

Prostanoid TP receptor stimulation enhances contractile activities in guinea pig urinary bladder smooth muscle through activation of Ca2+ entry channels: Potential targets in the treatment of urinary bladder contractile dysfunction

AimsWe examined the potential stimulatory effects of U46619 (a prostanoid TP receptor agonist) and five prostanoids on the contractile activities of urinary bladder smooth muscle (UBSM), focusing on the role of the TP receptor and its associated Ca2+ influx routes to understand the roles of prostanoids in the regulation of UB contractile activity. Main methodsChanges in the basal tone and spontaneous contractile activity (amplitude and frequency) of isolated guinea pig UBSM were measured isotonically. The presence of TP receptors in UBSM was examined by RT-qPCR and immunofluorescence. Key findingsU46619, prostaglandin (PG) E2, PGF2α, and PGA2 enhanced UBSM basal tone and spontaneous contractile activities, which were measured as amplitudes and frequencies. The enhancing effects of U46619 were completely suppressed by SQ 29,548 (a TP receptor antagonist), which also partially suppressed the stimulating effects of other prostanoids. The expression of TP receptors in UBSMs was verified at the mRNA and protein level. The enhancing effects of U46619 completely disappeared in Ca2+-free solution. U46619-enhanced basal tone was completely suppressed by verapamil, an inhibitor of voltage-dependent Ca2+ channels (VDCCs), and verapamil strongly decreased the spontaneous contraction frequency. The spontaneous contractions remaining in the presence of verapamil were strongly suppressed by SKF-96365 (an inhibitor of receptor-operated Ca2+ channels (ROCCs)/store-operated Ca2+ channels (SOCCs)), but not by LOE-908 (an inhibitor of ROCCs). SignificanceProstanoids can enhance UBSM contractile activities and thus may be endogenous candidates for induction of detrusor overactivity. The TP receptor and TP-receptor-activated VDCCs/SOCCs are key molecules responsible for these effects.

Urothelial Calcium-Sensing Receptor Modulates Micturition Function via Mediating Detrusor Activity and Ameliorates Bladder Hyperactivity in Rats.

The urothelium displays mechano- and chemosensory functions via numerous receptors and channels. The calcium-sensing receptor (CaSR) detects extracellular calcium and modulates several physiological functions. Nonetheless, information about the expression and the role of CaSR in lower urinary tract has been absent. We aimed to determine the existence of urothelial CaSR in urinary bladder and its effect on micturition function. We utilized Western blot to confirm the expression of CaSR in bladder and used immunofluorescence to verify the location of the CaSR in the bladder urothelium via colocalization with uroplakin III A. The activation of urothelial CaSR via the CaSR agonist, AC-265347 (AC), decreased urinary bladder smooth muscle (detrusor) activity, whereas its inhibition via the CaSR antagonist, NPS-2143 hydrochloride (NPS), increased detrusor activity in in vitro myography experiments. Cystometry, bladder nerve activities recording, and bladder surface microcirculation detection were conducted to evaluate the effects of the urothelial CaSR via intravesical administrations. Intravesical AC inhibited micturition reflex, bladder afferent and efferent nerve activities, and reversed cystitis-induced bladder hyperactivity. The urothelial CaSR demonstrated a chemosensory function, and modulated micturition reflex via regulating detrusor activity. This study provided further evidence of how the urothelial CaSR mediated micturition and implicated the urothelial CaSR as a potential pharmacotherapeutic target in the intervention of bladder disorders.

Inhibitory Effects of Antipsychotics on the Contractile Response to Acetylcholine in Rat Urinary Bladder Smooth Muscles.

The clinical applications of antipsychotics for symptoms unrelated to schizophrenia, such as behavioral and psychological symptoms, in patients with Alzheimer's disease, and the likelihood of doctors prescribing antipsychotics for elderly people are increasing. In elderly people, drug-induced and aging-associated urinary disorders are likely to occur. The most significant factor causing drug-induced urinary disorders is a decrease in urinary bladder smooth muscle (UBSM) contraction induced by the anticholinergic action of therapeutics. However, the anticholinergic action-associated inhibitory effects of antipsychotics on UBSM contraction have not been sufficiently assessed. In this study, we examined 26 clinically available antipsychotics to determine the extent to which they inhibit acetylcholine (ACh)-induced contraction in rat UBSM to predict the drugs that should not be used by elderly people to avoid urinary disorders. Of the 26 antipsychotics, six (chlorpromazine, levomepromazine (phenothiazines), zotepine (a thiepine), olanzapine, quetiapine, clozapine (multi-acting receptor targeted antipsychotics (MARTAs))) competitively inhibited ACh-induced contractions at concentrations corresponding to clinically significant doses. Further, 11 antipsychotics (perphenazine, fluphenazine, prochlorperazine (phenothiazines), haloperidol, bromperidol, timiperone, spiperone (butyrophenones), pimozide (a diphenylbutylpiperidine), perospirone, blonanserin (serotonin-dopamine antagonists; SDAs), and asenapine (a MARTA)) significantly suppressed ACh-induced contraction; however, suppression occurred at concentrations substantially exceeding clinically achievable blood levels. The remaining nine antipsychotics (pipamperone (a butyrophenone), sulpiride, sultopride, tiapride, nemonapride (benzamides), risperidone, paliperidone (SDAs), aripiprazole, and brexpiprazole (dopamine partial agonists)) did not inhibit ACh-induced contractions at concentrations up to 10-5 M. These findings suggest that chlorpromazine, levomepromazine, zotepine, olanzapine, quetiapine, and clozapine should be avoided by elderly people with urinary disorders.

Caldesmon ablation in mice causes umbilical herniation and alters contractility of fetal urinary bladder smooth muscle.

The actin-, myosin-, and calmodulin-binding protein caldesmon (CaD) is expressed in two splice isoforms: h-CaD, which is an integral part of the actomyosin domain of smooth muscle cells, and l-CaD, which is widely expressed and is involved in many cellular functions. Despite extensive research for many years, CaD's in vivo function has remained elusive. To explore the role of CaD in smooth muscle contraction in vivo, we generated a mutant allele that ablates both isoforms. Heterozygous animals were viable and had a normal life span, but homozygous mutants died perinatally, likely because of a persistent umbilical hernia. The herniation was associated with hypoplastic and dysmorphic abdominal wall muscles. We assessed mechanical parameters in isometrically mounted longitudinal strips of E18.5 urinary bladders and in ring preparations from abdominal aorta using wire myography. Ca2+ sensitivity was higher and relaxation rate was slower in Cald1-/- compared with Cald1+/+ skinned bladder strips. However, we observed no change in the content and phosphorylation of regulatory proteins of the contractile apparatus and myosin isoforms known to affect these contractile parameters. Intact fibers showed no difference in actin and myosin content, regardless of genotype, although KCl-induced force tended to be lower in homozygous and higher in heterozygous mutants than in WTs. Conversely, in skinned fibers, myosin content and maximal force were significantly lower in Cald1-/- than in WTs. In KO abdominal aortas, resting and U46619 elicited force were lower than in WTs. Our results are consistent with the notion that CaD impacts smooth muscle function dually by (1) acting as a molecular brake on contraction and (2) maintaining the structural integrity of the contractile machinery. Most importantly, CaD is essential for resolution of the physiological umbilical hernia and ventral body wall closure.

K V 7 Channel Subtype Expression and Physiological Roles in Rat Urinary Bladder Smooth Muscle Excitability and Contractility

Introduction Voltage-gated K+ (KV) channels, specifically type 7 (KV7), are emerging key regulators of urinary bladder smooth muscle (UBSM) excitability and contractility. The KV7 channel family consists of 5 pore-forming α-subunits (KV7.1–KV7.5) capable of assembling as homotetrameric (e.g. KV7.4) or heterotetrameric (e.g. KV7.4/KV7.5) channels; thus, further increasing species/tissue expression diversity and regulatory potential. However, much remains unknown about KV7 channel properties in UBSM and the translational value of the common experimental animal model - the rat. Hypothesis KV7 channels, especially homomeric or heteromeric KV7.4 and KV7.5 subtypes, regulate rat UBSM excitability and contractility. Methods UBSM tissue without mucosa was obtained from adult male Sprague Dawley rats. UBSM homogenates were used for Western blots to detect KV7 channel subtypes with or without surface biotinylation. UBSM isometric tension recordings were carried out in the presence of tetrodotoxin for spontaneous, 20 mM K+-induced and 1 µM carbachol-induced contractions or in its absence for the electrical field stimulated (EFS) contractions. For electrophysiological amphotericin-B perforated patch-clamp studies, single UBSM cells were prepared using enzymatic dissociation. UBSM cell membrane potential was recorded in current-clamp (I=0) mode at room temperature. The following KV7 channel modulators were tested: XE991 (KV7.1-KV7.5 subtype blocker), retigabine (KV7.2-KV7.5 activator), and ICA-069673 (selective activator for KV7.2/KV7.3, which at higher concentrations preferentially enhances KV7.4 and KV7.4/KV7.5 but not KV7.5 channels). Results Western blot experiments detected KV7.4 and KV7.5 channels in UBSM under both non- and surface-biotinylation assay conditions. The latter condition revealed that for both KV7.4 and KV7.5 subtypes surface localization predominated (>75 %). In isometric tension experiments, retigabine attenuated contractility in a concentration-dependent manner for all four contraction protocols (EC50 values: 2.6-58.7 µM for the amplitude and force, with complete inhibition of contractility at 100 µM, n=3-10). XE991 (10 µM) depolarized the membrane potential of isolated UBSM cells (Δ10.5±3.6 mV, n=5, p<0.05). In contrast, retigabine (10 µM, Δ-6.5±1.8 mV, n=4, p<0.04) and ICA-069673 (10 µM, Δ-11.5±2.4 mV, n=4, p<0.02), when applied separately, induced UBSM cell hyperpolarization. In the presence of XE991, ICA-069673 (10 µM) did not significantly change the membrane potential of isolated UBSM cells (Δ0.4±1.2 mV, n=4, p>0.05). Conclusions Our data show that both KV7.4 and KV7.5 channel subtypes are expressed in rat UBSM and display preferential cell membrane localization. Pharmacological modulation of KV7 channels can control UBSM cell excitability and tissue contractility. The hyperpolarizing effect of ICA-069673, which is blocked by XE991, revealed a critical role of KV7.4 homomeric and/or KV7.4/KV7.5 heteromeric channels in UBSM function.

The mast cell activator Compound 48/80 causes phasic urinary bladder smooth muscle contractions independent of histamine release

Inflammatory mediators released from mast cells cause both transient and phasic contractions of urinary bladder smooth muscle (UBSM). These contractions are viewed as initial disruptors of normal bladder function and can lead to profound bladder pathologies. However, the mechanisms responsible for each of these contractions has not been thoroughly investigated. Previously, we discovered that histamine-mediated contractions rapidly desensitize, whereas the mast cell activator compound 48/80 (10 µg/ml) caused both a long-lasting increase in the amplitude and frequency of phasic contractions in UBSM strips as well as a transient increase in baseline tension. Thus, we hypothesized that phasic contractions caused by compound 48/80 depended on the release of histamine from mast cells. Isometric contractility was performed with urothelium-intact UBSM strips from C57BL/6 mice in the presence or absence of the following drugs: the cholinergic antagonist atropine (2 µM); the 5HT2A receptor antagonist MDL 11939 (1 µM); the histamine H1 receptor antagonist fexofenadine (10 µM), the histamine H2 receptor antagonist roxatidine (25 µM), the H2 antagonist cimetidine (10 µM); or the mast cell stabilizer cromolyn (100 µM). None of the compounds tested significantly reduced the amplitude and duration of the spontaneous phasic contractions caused by compound 48/80. However, both MDL 11939 and cromolyn significantly reduced the baseline contraction. Our data suggest that responses to the mast cell activator compound 48/80 are a superimposition of 2 components: (1) a slowly desensitizing increase in baseline tension caused by release of 5-HT from mast cells, and (2) large-amplitude phasic contractions that are histamine- and 5HT-independent. However, the mechanism responsible for increases in the amplitude of phasic contractions remains unclear.

Mechanism of the Long-lasting Potentiating Effect of Distigmine on Urinary Bladder Motility

Distigmine bromide (distigmine) is a carbamate cholinesterase (ChE) inhibitor, which is mainly used for the treatment of myasthenia gravis. Distigmine is also used in Japan for the treatment for underactive bladder and glaucoma. The effectiveness of distigmine for underactive bladder treatment has been confirmed by many clinical reports, and this effect is thought to be caused by potentiating urinary bladder smooth muscle contraction due to inhibition of acetylcholine degradation during micturition. However, the pharmacological effects of distigmine on urinary bladder smooth muscle have not been well studied. The most distinctive pharmacological feature of distigmine is that it shows long-lasting effects than other ChE inhibitors; however, few studies have investigated the persistence of the enhancing effect of distigmine on the contractile function of urinary bladder smooth muscle. Moreover, this mechanism remains unclear. In this review, we present our findings on the mechanism of the potentiating effect of distigmine on isolated guinea pig urinary bladder smooth muscle contraction. We also discuss the long-lasting potentiating effect of distigmine on urinary bladder motility and the mechanism of these effects using guinea pig urinary bladder smooth muscle in vivo and in vitro. In addition, we present our investigations on the long-lasting mechanism of distigmine using recombinant human acetylcholinesterase.

Identification and Characterization of a Novel Large-Conductance Calcium-Activated Potassium Channel Activator, CTIBD, and Its Relaxation Effect on Urinary Bladder Smooth Muscle.

The large-conductance calcium-activated potassium channel (BKCa channel) is expressed on various tissues and is involved in smooth muscle relaxation. The channel is highly expressed on urinary bladder smooth muscle cells and regulates the repolarization phase of the spontaneous action potentials that control muscle contraction. To discover novel chemical activators of the BKCa channel, we screened a chemical library containing 8364 chemical compounds using a cell-based fluorescence assay. A chemical compound containing an isoxazolyl benzene skeleton (compound 1) was identified as a potent activator of the BKCa channel and was structurally optimized through a structure-activity relationship study to obtain 4-(4-(4-chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol (CTIBD). When CTIBD was applied to the treated extracellular side of the channel, the conductance-voltage relationship of the channel shifted toward a negative value, and the maximum conductance increased in a concentration-dependent manner. CTIBD altered the gating kinetics of the channel by dramatically slowing channel closing without effecting channel opening. The effects of CTIBD on bladder muscle relaxation and micturition function were tested in rat tissue and in vivo. CTIBD concentration-dependently reduced acetylcholine-induced contraction of urinary bladder smooth muscle strips. In an acetic acid-induced overactive bladder (OAB) model, intraperitoneal injection of 20 mg/kg CTIBD effectively restored frequent voiding contraction and lowered voiding volume without affecting other bladder function parameters. Thus, our results indicate that CTIBD and its derivatives are novel chemical activators of the bladder BKCa channel and potential candidates for OAB therapeutics. SIGNIFICANCE STATEMENT: The novel BKCa channel activator CTIBD was identified and characterized in this study. CTIBD directly activates the BKCa channel and relaxes urinary bladder smooth muscle of rat, so CTIBD can be a potential candidate for overactive bladder therapeutics.

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.

Urinary bladder smooth muscle ion channels: expression, function, and regulation in health and disease.

Urinary bladder smooth muscle (UBSM), also known as detrusor smooth muscle, forms the bladder wall and ultimately determines the two main attributes of the organ: urine storage and voiding. The two functions are facilitated by UBSM relaxation and contraction, respectively, which depend on UBSM excitability shaped by multiple ion channels. In this review, we summarize the current understanding of key ion channels establishing and regulating UBSM excitability and contractility. They include excitation-enhancing voltage-gated Ca2+ (Cav) and transient receptor potential channels, excitation-reducing K+ channels, and still poorly understood Cl- channels. Dynamic interplay among UBSM ion channels determines the overall level of Cav channel activity. The net Ca2+ influx via Cav channels increases global intracellular Ca2+ concentration, which subsequently triggers UBSM contractility. Here, for each ion channel type, we describe UBSM tissue/cell expression (mRNA and protein) profiles and their role in regulating excitability and contractility of UBSM in various animal species, including the mouse, rat, and guinea pig, and, most importantly, humans. The currently available data reveal certain interspecies differences, which complicate the translational value of published animal research results to humans. This review highlights recent developments, findings on genetic knockout models, pharmacological data, reports on UBSM ion channel dysfunction in animal bladder disease models, and the very limited human studies currently available. Among all gaps in present-day knowledge, the unknowns on expression and functional roles for ion channels determined directly in human UBSM tissues and cells under both normal and disease conditions remain key hurdles in the field.

Poly-phosphate increases SMC differentiation of mesenchymal stem cells on PLGA-polyurethane nanofibrous scaffold.

The use of bioactive scaffolds in tissue engineering has a significant effect on the damaged tissue healing by an increase in speed and quality of the process. Herein, electrospinning was applied to fabricate composite nanofibrous scaffolds by Poly lactic-co-glycolic acid (PLGA) and Polyurethane (PU) with and without poly-phosphate (poly-P). Scaffolds were characterized morphologically by scanning electron microscope (SEM), and their biocompatibility was also investigated by SEM, protein adsorption, cell attachment and survival assays. The applicability of the scaffolds for bladder tissue engineering was also evaluated by culturing mesenchymal stem cells (MSCs) on the scaffolds and their differentiation into smooth muscle cell (SMC) was studied at the gene and protein levels. The results demonstrated that scaffold biocompatibility was increased significantly by loading poly-P. SMC related gene and protein expression level in MSCs cultured on poly-P-loaded scaffold was also increased significantly compared to those cells cultured on empty scaffold. It can be concluded that poly-P hasn’t also increased scaffold biocompatibility, but also SMC differentiation potential of MSCs was also increased while cultured on the poly-P containing scaffold compared to the empty scaffold. Taken together, our study showed that PLGA–PU–poly-P alone and in combination with MSCs has a promising potential for support urinary bladder smooth muscle tissue engineering.

Histamine may directly contract urinary bladder smooth muscle

Mast cell degranulation and histamine release contribute to painful bladder syndrome, urinary tract infections, interstitial cystitis and other bladder disorders. The effects of histamine are well‐studied in other tissues, but little is known about the role of histamine and histamine receptors within the bladder. Since other smooth muscle cells express H1 (contractile) and H2 (relaxant) histamine receptors, we tested the hypothesis that histamine causes contraction of the urinary bladder smooth muscle in mice. All procedures followed institutional guidelines and were approved by the Institutional Animal Care and Use Committees of Michigan State University. Six to 12‐week‐old C57Bl/6 mice were euthanized and dissected bladders were cut into approximately 2 mm wide bladder strips, with or without the urothelium attached, for isometric contractility experiments. Both histamine H1 and H2 receptors were expressed in bladder smooth muscle cells. Increasing concentrations of histamine (100 nM – 300 μM) did not contract strips without urothelium and minimally contracted bladder strips with urothelium. However, bolus administration of 200 μM histamine caused a rapid but transient contraction in strips with and without urothelium. The H1 receptor blocker fexofenadine (5 – 10 μM) blocked these contractions (P&lt;0.05; N=5), whereas the H2 receptor blocker cimetidine (5 – 10 μM) had no effect. In addition, transient receptor potential vanilloid 1 (TRPV1) channel blocker capsazepine significantly reduced histamine‐induced contractions only in bladder strips without urothelium, whereas the Na+ channel blocker tetrodotoxin (TTX) had no effect in any tissue (P &lt; 0.05; N= 3–5). The mast cell activator compound 48/80 (10 μg/ml) contracted urinary bladder smooth muscle, and this contraction was also unaffected by TTX. These results indicate that H1 receptors on urinary bladder smooth cells likely modulate histamine‐induced bladder contraction, and sufficient amounts of histamine can be released from mast cells to cause a contraction.Support or Funding InformationThis research is supported by NIH K01‐DK103840 (NRT) and NIH R01‐DK119615 (NRT &amp; GCM).

Distinct Age‐dependent Effects of Glibenclamide on Guinea Pig Urinary Bladder Smooth Muscle Non‐selective Cation Currents and Spontaneous Phasic Contractions

Glibenclamide blocks ATP‐sensitive K+ channels (Kir6.x‐SUR) via sulphonylurea receptor (SUR) subunit engagement. Glibenclamide can also inhibit TRPM4‐SUR channel complexes via SUR interaction or TRPM4 channels directly. Recently, our group has found that glibenclamide inhibits whole‐cell non‐selective cation (NSC) currents and spontaneous phasic contractions of guinea pig urinary bladder smooth muscle (UBSM) cells and isolated tissue strips, respectively, although less effectively than 9‐phenanthrol, a TRPM4 channel inhibitor. Here, the age‐dependent effects of glibenclamide and 9‐phenanthrol were determined on guinea pig UBSM whole‐cell NSC currents and spontaneous phasic contractions. Urinary bladders were obtained from young (5–8 wk old, 350–650 g) and retired breeder (RB, 0.5–1.5 yr old, &gt;800 g) male Hartley guinea pigs (Charles River). Mucosa was removed and UBSM strips prepared for isometric tension recordings (spontaneous phasic contractions) or for subsequent enzymatic treatment yielding freshly isolated UBSM cells. UBSM cells were then used in amphotericin‐perforated patch‐clamp recordings either (1) whole‐cell NSC currents optimized by blocking K+ and Ca2+ selective currents or (2) cell membrane potential (I=0) with K+ and Ca2+ channel currents intact. Glibenclamide (100 μM) and 9‐phenanthrol (100 μM) inhibited the voltage‐step‐induced NSC currents in RB‐group UBSM cells; at +106 mV, the respective decreases were 36.6±4.8% (n=6, p&lt;0.0001) and 42.9±3.9% (n=9, p&lt;0.001). In UBSM cells of young guinea pigs, 9‐phenanthrol (100 μM, at +106 mV, 50.4±2.9%, n=7, p&lt;0.001) — but not glibenclamide (100 μM, at +106 mV, 4.6±8.3%, n=6) — caused attenuation in the whole‐cell NSC currents. Glibenclamide (100 μM) did not change the membrane potential in UBSM cells of either young (control: −27.8±4.0 mV; change: +4.2±2.4 mV, n=9, p=0.11) or RB (control: −11.6±4.0 mV; change: −0.42±0.8 mV, n=4, p=0.65) guinea pigs. Of note, a majority of UBSM cells of young (5/9 cells), but not RB‐group (0/4 cells), responded to glibenclamide with depolarization (&gt;3 mV). Spontaneous phasic contractions were concentration‐dependently inhibited by glibenclamide and 9‐phenanthrol in UBSM strips of both age groups (n=5–10). Only glibenclamide displayed age‐dependent differential effects on the muscle force (ANOVA, p=0.04), and the phasic contraction amplitude just failed to reach the statistical significance (p=0.07). IC50 values of glibenclamide for the two parameters were 2–3‐fold more potent in young‐ (~14, 15 μM) than RB‐group UBSM strips (31, 45 μM) with high maximum inhibitions (72–93%, n=7–10). This supports the concept that in RB guinea pigs glibenclamide‐sensitive NSC currents oppose UBSM relaxation. In summary, glibenclamide but not 9‐phenanthrol exhibits age‐dependent effects on whole‐cell NSC currents and spontaneous phasic contractions. In old but not young guinea pigs, glibenclamide‐sensitive NSC channels may contribute to the regulation of UBSM excitability and contractility.Support or Funding InformationNIH R01‐DK106964 and P20‐DK123971 to Georgi V. Petkov.

A Method for Preparation of Freshly-Isolated Human Urinary Bladder Smooth Muscle Cells: Utility for Characterization of Whole-Cell Cation Currents

Ion channels of the urinary bladder smooth muscle (UBSM) determine cellular excitability and contractility and, hence, regulate the two main functions of the organ, urine storage and voiding. Preparation of the viable, fresh, and single UBSM cells presents an important technical challenge for subsequent biophysical and electrophysiological studies. Here, we describe a method for successfully obtaining freshly-isolated human single UBSM cells from patient-donors undergoing open bladder surgeries and its application for the recording of voltage-step-induced cation currents. The initial step in the method involves dissection to separate the UBSM layer (also known as detrusor smooth muscle and muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa). The UBSM is then cut into small pieces in Ca2+-free (nominal) dissection/digestion solution (DS). UBSM pieces are next transferred to and sequentially treated separately with DS containing papain and collagenase at ∼37 °C for 30-45 min per step. Following washes with enzyme-free DS and trituration with a fire-polished pipette, the pieces release single UBSM cells. Freshly-isolated UBSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations. Specifically, single UBSM cells contract in response to the muscarinic agonist carbachol (3 μM). In amphotericin-B perforated patch-clamp recordings, the TRPM4 channel blocker 9-phenanthrol concentration-dependently reduces voltage-step-induced cation currents. UBSM cells can also be studied by other techniques such as: single cell RT-PCR, microarray analysis, immunocytochemistry, in situ proximity ligation assay, and Ca2+ imaging. Studies on freshly-isolated human UBSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable channels in the urinary bladder and will continue as a gold standard in elucidating UBSM cellular properties and regulatory mechanisms. Funding: NIH R01DK106964 grant to Georgi V. Petkov.

A novel contraction mechanism by intracellular GDP in bladder smooth muscle

Background: When we tried to elucidate the mechanism of cooling induced contraction using pig urinary bladder smooth muscle, we found the tension force was increased by intracellular GDP with dose dependent manner.

NK2 receptor-mediated detrusor muscle contraction involves Gq/11-dependent activation of voltage-dependent Ca2+ channels and the RhoA-Rho kinase pathway.

Tachykinins (TKs) are involved in both the physiological regulation of urinary bladder functions and development of overactive bladder syndrome. The aim of the present study was to investigate the signal transduction pathways of TKs in the detrusor muscle to provide potential pharmacological targets for the treatment of bladder dysfunctions related to enhanced TK production. Contraction force, intracellular Ca2+ concentration, and RhoA activity were measured in the mouse urinary bladder smooth muscle (UBSM). TKs and the NK2 receptor (NK2R)-specific agonist [β-Ala8]-NKA(4-10) evoked contraction, which was inhibited by the NKR2 antagonist MEN10376. In Gαq/11-deficient mice, [β-Ala8]-NKA(4-10)-induced contraction and the intracellular Ca2+ concentration increase were abolished. Although Gq/11 proteins are linked principally to phospholipase Cβ and inositol trisphosphate-mediated Ca2+ release from intracellular stores, we found that phospholipase Cβ inhibition and sarcoplasmic reticulum Ca2+ depletion failed to have any effect on contraction induced by [β-Ala8]-NKA(4-10). In contrast, lack of extracellular Ca2+ or blockade of voltage-dependent Ca2+ channels (VDCCs) suppressed contraction. Furthermore, [β-Ala8]-NKA(4-10) increased RhoA activity in the UBSM in a Gq/11-dependent manner and inhibition of Rho kinase with Y-27632 decreased contraction force, whereas the combination of Y-27632 with either VDCC blockade or depletion of extracellular Ca2+ resulted in complete inhibition of [β-Ala8]-NKA(4-10)-induced contractions. In summary, our results indicate that NK2Rs are linked exclusively to Gq/11 proteins in the UBSM and that the intracellular signaling involves the simultaneous activation of VDCC and the RhoA-Rho kinase pathway. These findings may help to identify potential therapeutic targets of bladder dysfunctions related to upregulation of TKs.

Effects of Distigmine on the Mechanical Activity of Urinary Bladder Smooth Muscle.

Distigmine bromide (distigmine) is a reversible carbamate cholinesterase (ChE) inhibitor. Its principle clinical application is in the treatment of myasthenia gravis. Distigmine is also used as a remedy for dysuria and glaucoma. Its effectiveness in the management of dysuria has been demonstrated in several clinical reports. Distigmine may improve (enhance) urinary bladder smooth muscle (UBSM) contraction during micturition by inhibiting acetylcholine (ACh) decomposition. However, the pharmacological effects of distigmine on UBSM have not been adequately studied so far. In this review article, we summarize the reported effects of distigmine on the contractile responses elicited by exogenous and endogenous ACh in isolated UBSM preparations. We also discuss the effects of distigmine on the UBSM basal tone and the contractile response of UBSM to ATP, which is co-released with ACh from parasympathetic nerve terminals.

Noradrenaline-Induced Relaxation of Urinary Bladder Smooth Muscle Is Primarily Triggered through the β3-Adrenoceptor in Rats.

β-Adrenoceptors are subclassified into 3 subtypes (β1-β3). Among these, β3-adrenoceptors are present in various types of smooth muscle and are believed to play a role in relaxation responses of these muscles. β3-Adrenoceptors are also present in urinary bladder smooth muscle (UBSM), although their expression varies depending on the animal species. To date, there has been little information available about the endogenous ligand that stimulates β3-adrenoceptors to produce relaxation responses in UBSM. In this study, to determine whether noradrenaline is a ligand of UBSM β3-adrenoceptors, noradrenaline-induced relaxation was analyzed pharmacologically using rat UBSM. We also assessed whether noradrenaline metabolites were ligands in UBSM. In isolated rat urinary bladder tissues, mRNAs for β1-, β2-, and β3-adrenoceptors were detected using RT-PCR. In UBSM preparations contracted with methacholine (3 × 10-5 M), noradrenaline-induced relaxation was not inhibited by the following antagonists: atenolol (10-6 M; selective β1-adrenoceptor antagonist), ICI-118,551 (3 × 10-8 M; selective β2-adrenoceptor antagonist), propranolol (10-7 M; non-selective β-adrenoceptor antagonist), and bupranolol (10-7 M; non-selective β-adrenoceptor antagonist). In the presence of propranolol (10-6 M), noradrenaline-induced relaxation was competitively inhibited by bupranolol (3 × 10-7-3 × 10-6 M) or SR59230A (10-7-10-6 M; selective β3-adrenoceptor antagonist), with their pA2 values calculated to be 6.64 and 7.27, respectively. None of the six noradrenaline metabolites produced significant relaxation of methacholine-contracted UBSM. These findings suggest that noradrenaline, but not its metabolites, is a ligand for β3-adrenoceptors to produce relaxation responses of UBSM in rats.

116 - Thromboxane prostanoid receptor signaling in the urinary bladder smooth muscle

116 - Thromboxane prostanoid receptor signaling in the urinary bladder smooth muscle

Phasic, Rapidly‐Propagating Calcium Transients Occur in a Non‐Muscle Cell Network within the Mouse Urinary Bladder Wall

Multiple species, including humans, exhibit “non‐voiding” or “transient” contractions of the bladder wall during filling that are accompanied by large bursts of activity from bladder sensory nerves. This suggests that transient contractions play an important role as sensors of bladder fullness, but how transient contractions are generated remains unclear. Using genetically‐encoded Ca2+ indicator mice and novel macroscopic imaging techniques, we sought to determine what Ca2+ signals drive TC generation and coordination across the bladder wall. We utilized mice that constitutively express the Ca2+ indicators GCaMP5 or GCaMP6f as an expression‐dependent fate map of smooth muscle cells (Acta2 promoter) or non‐muscle cells (Trpv1 promoter) within the urinary bladder wall. We discovered that transient pressure events (TPEs) are actually the integration of numerous micro‐contractions of the bladder wall. These micromotions vary in position, frequency and duration, but result in regular, phasic increases in intravesical pressure. Ca2+ events in urinary bladder smooth muscle (UBSM) were restricted to small patches muscle cells and seemed to occur stochastically. Interestingly, we discovered a network of cells within the bladder wall of Trpv1‐GCaMP6f mice that were neither smooth muscle cells nor nerve fibers. This network exhibited extremely rapid and phasic Ca2+ transients that propagated quickly across the entire bladder wall, suggesting that the cells were electrically coupled. The slower waxing and waning of the high‐frequency Ca2+ transient intensity closely aligned with the frequency of TPEs. Surprisingly, this network of cells was unresponsive to the TRPV1 agonist capsaicin, suggesting that TRPV1 channels were no longer functional in these cells. These data suggest that the bladder wall micro‐motions underlying TPEs may be driven by phasic Ca2+ transients that occur in a non‐muscle cell network within the bladder wall.Support or Funding InformationSupported by NIH K01DK103840 (NRT) and R37DK053832 (MTN).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.