Urinary Bladder Smooth Muscle Research Articles

Assessment of Inhibitory Effects of Hypnotics on Acetylcholine-Induced Contractions in Isolated Rat Urinary Bladder Smooth Muscle

The present study aimed to investigate the potential inhibitory effects of 21 clinically available hypnotics on acetylcholine (ACh)-induced contractions in rat urinary bladder smooth muscle (UBSM) in order to predict whether these hypnotics could induce voiding impairment. ACh-induced contraction in rat UBSM was inhibited only by diphenhydramine (a histamine H1 receptor antagonist) at a concentration that was clinically relevant. ACh-induced contraction was also significantly inhibited by flurazepam (a benzodiazepine hypnotic) and suvorexant (an orexin receptor antagonist), albeit at concentrations that substantially exceeded clinically achievable blood levels. These three drugs (at 10-5 M) also inhibited high-KCl (80 mM) Locke-Ringer solution-induced contractions. In contrast to the effects of the abovementioned hypnotics, ACh-induced contractions were not significantly affected by triazolam, etizolam, brotizolam, lormetazepam, estazolam, flunitrazepam, nitrazepam (benzodiazepine hypnotics), thiopental, thiamylal, pentobarbital, amobarbital, secobarbital, phenobarbital (barbiturate hypnotics), zolpidem (an imidazopyridine hypnotic), zopiclone (a cyclopyrrolone hypnotic), ramelteon (a melatonin receptor agonist), bromovalerylurea, and chloral hydrate. These findings suggest that most clinically used hypnotics are not likely to result in anticholinergic-induced dysuria within their clinically achievable blood concentration ranges. Diphenhydramine may, however, induce voiding impairment, an action attributable to diminished UBSM contractility within its clinical dose range.

Ketamine‑induced bladder dysfunction is associated with extracellular matrix accumulation and impairment of calcium signaling in a mouse model.

Due to the rising abuse of ketamine usage in recent years, ketamine-induced urinary tract syndrome has received increasing attention. The present study aimed to investigate the molecular mechanism underlying ketamine-associated cystitis in a mouse model. Female C57BL/6 mice were randomly divided into two groups: One group was treated with ketamine (100 mg/kg/day of ketamine for 20 weeks), whereas, the control group was treated with saline solution. In each group, micturition frequency and urine volume were examined to assess urinary voiding functions. Mouse bladders were extracted and samples were examined for pathological and morphological alterations using hematoxylin and eosin staining, Masson's trichrome staining and scanning electron microscopy. A cDNA microarray was conducted to investigate the differentially expressed genes following treatment with ketamine. The results suggested that bladder hyperactivity increased in the mice treated with ketamine. Furthermore, treatment with ketamine resulted in a smooth apical epithelial surface, subepithelial vascular congestion and lymphoplasmacytic aggregation. Microarray analysis identified a number of genes involved in extracellular matrix accumulation, which is associated with connective tissue fibrosis progression, and in calcium signaling regulation, that was associated with urinary bladder smooth muscle contraction. Collectively, the present results suggested that these differentially expressed genes may serve critical roles in ketamine-induced alterations of micturition patterns and urothelial pathogenesis. Furthermore, the present findings may provide a theoretical basis for the development of effective therapies to treat ketamine-induced urinary tract syndrome.

Assessment of the inhibitory effects of antipsychotics on acetylcholine (ACh)-induced contraction in rat urinary bladder smooth muscle (UBSM)

Assessment of the inhibitory effects of antipsychotics on acetylcholine (ACh)-induced contraction in rat urinary bladder smooth muscle (UBSM)

The KV 7 channel activator retigabine suppresses mouse urinary bladder afferent nerve activity without affecting detrusor smooth muscle K+ channel currents.

KV 7 channels are a family of voltage-dependent K+ channels expressed in many cell types, which open in response to membrane depolarization to regulate cell excitability. Drugs that target KV 7 channels are used clinically to treat epilepsy. Interestingly, these drugs also cause urinary retention, but it was unclear how. In this study, we focused on two possible mechanisms by which retigabine could cause urinary retention: by decreasing smooth muscle excitability, or by decreasing sensory nerve outflow. Urinary bladder smooth muscle had no measurable KV 7 channel currents. However, the KV 7 channel agonist retigabine nearly abolished sensory nerve outflow from the urinary bladder during bladder filling. We conclude that KV 7 channel activation likely affects urinary bladder function by blocking afferent nerve outflow to the brain, which is key to sensing bladder fullness. KV 7 channels are voltage-dependent K+ channels that open in response to membrane depolarization to regulate cell excitability. KV 7 activators, such as retigabine, were used to treat epilepsy but caused urinary retention. Using electrophysiological recordings from freshly isolated mouse urinary bladder smooth muscle (UBSM) cells, isometric contractility of bladder strips, and ex vivo measurements of bladder afferent activity, we explored the role of KV 7 channels as regulators of murine urinary bladder function. The KV 7 activator retigabine (10μM) had no effect on voltage-dependent K+ currents or resting membrane potential of UBSM cells, suggesting that these cells lacked retigabine-sensitive KV 7 channels. The KV 7 inhibitor XE-991 (10μM) inhibited UBSM K+ currents; the properties of these currents, however, were typical of KV 2 channels and not KV 7 channels. Retigabine inhibited voltage-dependent Ca2+ channel (VDCC) currents and reduced steady-state contractions to 60mM KCl in bladder strips, suggesting that reduction in VDCC current was sufficient to directly affect UBSM function. To determine if retigabine altered ex vivo bladder sensory outflow, we measured afferent activity during simulated transient contractions (TCs) of the bladder wall. Simulated TCs caused bursts of afferent activity that were nearly abolished by retigabine. The effects of retigabine were blocked by co-incubation with XE-991, suggesting specific activation of KV 7 channels on afferent nerves. These results indicate that retigabine primarily affects urinary bladder function by inhibiting TC generation and afferent nerve activity, which are key to sensing bladder fullness. Any direct inhibition of UBSM contractility is likely to be from non-specific effects on VDCCs and KV 2 channels.

A biophysically constrained computational model of the action potential of mouse urinary bladder smooth muscle.

Urinary incontinence is associated with enhanced spontaneous phasic contractions of the detrusor smooth muscle (DSM). Although a complete understanding of the etiology of these spontaneous contractions is not yet established, it is suggested that the spontaneously evoked action potentials (sAPs) in DSM cells initiate and modulate the contractions. In order to further our understanding of the ionic mechanisms underlying sAP generation, we present here a biophysically detailed computational model of a single DSM cell. First, we constructed mathematical models for nine ion channels found in DSM cells based on published experimental data: two voltage gated Ca2+ ion channels, an hyperpolarization-activated ion channel, two voltage-gated K+ ion channels, three Ca2+-activated K+ ion channels and a non-specific background leak ion channel. The ion channels’ kinetics were characterized in terms of maximal conductances and differential equations based on voltage or calcium-dependent activation and inactivation. All ion channel models were validated by comparing the simulated currents and current-voltage relations with those reported in experimental work. Incorporating these channels, our DSM model is capable of reproducing experimentally recorded spike-type sAPs of varying configurations, ranging from sAPs displaying after-hyperpolarizations to sAPs displaying after-depolarizations. The contributions of the principal ion channels to spike generation and configuration were also investigated as a means of mimicking the effects of selected pharmacological agents on DSM cell excitability. Additionally, the features of propagation of an AP along a length of electrically continuous smooth muscle tissue were investigated. To date, a biophysically detailed computational model does not exist for DSM cells. Our model, constrained heavily by physiological data, provides a powerful tool to investigate the ionic mechanisms underlying the genesis of DSM electrical activity, which can further shed light on certain aspects of urinary bladder function and dysfunction.

Effect of pregnancy on cooling tone and rhythmic contractions of the rat urinary bladder.

Pregnancy is a physiological alteration that can affect urinary bladder. Cooling of urinary bladder smooth muscle is known as a potent stimulus to micturition due to an increase in muscle tone. The current study investigates the effects of pregnancy on cooling tone and on the rhythmic contractions of the urinary bladder. Twenty-four rats were used in this study as control group (non-pregnant) and pregnant group (18-20-day pregnancy). Isolated rat urinary muscle strips were suspended in organ baths containing Krebs' solution for isometric tension recording. Cooling from 37 to 5°C induced a rapid and reproducible increase in basal tone, proportional to cooling temperature. Cooling also increased the rhythmic activity (amplitude and frequency) at 30 and 25°C, then decreased at 20°C, and abolished at 15-5°C. These responses were more pronounced in pregnant group than in control group. Rhythmic contractions were abolished in calcium-free, EGTA (1mM)-containing Krebs' solution and in the presence of nifedipine, while they were not affected by CPA or TTX in both groups. Our investigation showed that the influx of extracellular calcium is important in inducing the rhythmic contractions. Pregnancy increases cooling-induced contraction in pregnant rat urinary preparations and its rhythmic contractions including amplitude and frequency than non-pregnant rat. Rhythmic contractions are myogenic in nature and highly extracellular calcium dependent. They may play a crucial role in urinary bladder overactivity and incontinence during pregnancy.

Signaling and metabolic properties of fast and slow smooth muscle types from mice

This study aims to improve the classification of smooth muscle types to better understand their normal and pathological functional phenotypes. Four different smooth muscle tissues (aorta, muscular arteries, intestine, urinary bladder) with a 5-fold difference in maximal shortening velocity were obtained from mice and classified according to expression of the inserted myosin heavy chain (SMHC-B). Western blotting and quantitative PCR analyses were used to determine 15 metabolic and 8 cell signaling key components in each tissue. The slow muscle type (aorta) with a 12 times lower SMHC-B had 6-fold lower expression of the phosphatase subunit MYPT1, a 7-fold higher expression of Rhokinase 1, and a 3-fold higher expression of the PKC target CPI17, compared to the faster (urinary bladder) smooth muscle. The slow muscle had higher expression of components involved in glucose uptake and glycolysis (type 1 glucose transporter, 3 times; hexokinase, 13 times) and in gluconeogenesis (phosphoenolpyruvate carboxykinase, 43 times), but lower expression of the metabolic sensing AMP-activated kinase, alpha 2 isoform (5 times). The slow type also had higher expression of enzymes involved in lipid metabolism (hormone-sensitive lipase, 10 times; lipoprotein lipase, 13 times; fatty acid synthase, 6 times; type 2 acetyl-coenzyme A carboxylase, 8 times). We present a refined division of smooth muscle into muscle types based on the analysis of contractile, metabolic, and signaling components. Slow compared to fast smooth muscle has a lower expression of the deactivating phosphatase and upregulated Ca2+ sensitizing pathways and is more adapted for sustained glucose and lipid metabolism.

Co-culturing porcine normal urothelial cells, urinary bladder fibroblasts and smooth muscle cells for tissue engineering research.

New strategies for culturing and co-culturing of the main types of urinary bladder cells are essential for successful establishment of biomimetic in vitro models, which could be applied for research into, and management of, diverse urological disorders. Porcine normal urothelial cells are available in nearly unlimited amounts and have many properties equivalent to human urothelial cells. In the present study, we established normal differentiated porcine urothelial cells in co-cultures with porcine urinary bladder normal fibroblasts and/or smooth muscle cells. The optimal culture medium for establishment of differentiated urothelial cells, demonstrated by positive immunofluorescence of uroplakins, cytokeratins (CK 7, CK 20), zonula occludens 1 (ZO-1), claudin 4, claudin 8, and E-cadherin, was the medium composed of equal parts of Advanced Dulbecco's modified Eagle's medium (A-DMEM) and MCDB 153 medium with physiological calcium concentration of 2.5 mM and without fetal bovine serum, named UroM (+Ca2+ - S). This medium was also proven to be suitable for culturing of bladder fibroblasts and smooth muscle cells and co-culturing of urothelial cells with these mesenchymal cells. Urothelial cell differentiation was optimal in UroM (+Ca2+ - S) medium in all co-culture conditions and when compared to all conditioned-media combinations. To summarize, these strategies for culturing and co-culturing of urinary bladder urothelial cells with mesenchymal cells could be used as new in vitro models for future basic and applicable research of the urinary bladder and thus potentially also for translational tissue engineering studies.

Does the Mesenchymal Stem Cell Source Influence Smooth Muscle Regeneration in Tissue-Engineered Urinary Bladders?

A variety of tissue engineering techniques utilizing different cells and biomaterials are currently being explored to construct urinary bladder walls de novo, but so far no approach is clearly superior. The aim of this study was to determine whether mesenchymal stem cells (MSCs) isolated from different sources, (bone marrow [BM-MSCs] and adipose tissue [ADSCs]), differ in their potential to regenerate smooth muscles in tissue-engineered urinary bladders and to determine an optimal number of MSCs for urinary bladder smooth muscle regeneration. Forty-eight rats underwent hemicystectomy and bladder augmentation with approximately 0.8 cm2 graft. In the first and second groups, urinary bladders were reconstructed with small intestinal submucosa (SIS) seeded with 10 × 106 or 4 × 106 ADSCs/cm2, respectively. In the third and fourth groups, urinary bladders were augmented with SIS seeded with 10 × 106 or 4 × 106 BM-MSCs/cm2, respectively. In the fifth group, urinary bladders were augmented with SIS without cells. The sixth group (control) was left intact. Smooth muscle regeneration was evaluated by real-time polymerase chain reaction (RT-PCR) and histological examinations. Histologically, there were no significant differences between urinary bladders augmented with ADSCs and BM-MSCs, but there was a marked increase in smooth muscle formation in bladders augmented with grafts seeded with MSCs in higher density (10 × 106/cm2) compared to lower density (4 × 106/cm2). Molecular analysis revealed that bladders reconstructed with ADSC-seeded grafts expressed higher levels of smooth muscle myosin heavy chain, caldesmon, and vinculin. Bladders augmented with unseeded SIS were fibrotic and devoid of smooth muscles. ADSCs and BM-MSCs have comparable smooth muscle regenerative potential, but the number of MSCs used for graft preparation significantly affects the smooth muscle content in tissue-engineered urinary bladders.

Local Ca2+ coupling between mitochondria and sarcoplasmic reticulum following depolarization in guinea pig urinary bladder smooth muscle cells.

Spatiotemporal changes in cytosolic Ca2+ concentration ([Ca2+]c) trigger a number of physiological functions in smooth muscle cells (SMCs). We previously imaged Ca2+-induced Ca2+ release following membrane depolarization as local Ca2+ transients, Ca2+ hotspots, in subplasmalemmal regions. In this study, the physiological significance of mitochondria on local Ca2+ signaling was examined. Cytosolic and mitochondrial Ca2+ images following depolarization or action potentials were recorded in single SMCs from the guinea pig urinary bladder using a fast-scanning confocal fluorescent microscope. Depolarization- and action potential-induced [Ca2+]c transients occurred at several discrete sites in subplasmalemmal regions, peaked within 30 ms, and then spread throughout the whole-cell. In contrast, Ca2+ concentration in the mitochondria matrix ([Ca2+]m) increased after a delay of ~50 ms from the start of depolarization, and then peaked within 500 ms. Following repolarization, [Ca2+]c returned to the resting level with a half-decay time of ~500 ms, while [Ca2+]m recovered more slowly (∼1.5 s). Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, a mitochondrial uncoupler, abolished depolarization-induced [Ca2+]m elevations and slowed [Ca2+]c changes. Importantly, short depolarization-induced changes in [Ca2+]m and transmembrane potential in mitochondria coupled to Ca2+ hotspots were significantly larger than those in other mitochondria. Total internal reflection fluorescence imaging revealed that a subset of mitochondria closely localized with ryanodine receptors and voltage-dependent Ca2+ channels. These results indicate that particular mitochondria are functionally coupled to ion channels and sarcoplasmic reticulum fragments within the local Ca2+ microdomain, and thus, strongly contribute to [Ca2+]c regulation in SMCs.

Ranatensin-HL: A Bombesin-Related Tridecapeptide from the Skin Secretion of the Broad-Folded Frog, Hylarana latouchii.

Bombesin-related peptides are a family of peptides whose prototype was discovered in amphibian skin and which exhibit a wide range of biological activities. Since the initial isolation of bombesin from Bombina bombina skin, diverse forms of bombesin-related peptides have been found in the skins across Anura. In this study, a novel bombesin-related peptide of the ranatensin subfamily, named ranatensin-HL, was structurally-characterised from the skin secretion of the broad-folded frog, Hylarana latouchii, through combination of molecular cloning and mass spectrometric methodologies. It is composed of 13 amino acid residues, pGlu-RAGNQWAIGHFM-NH2, and resembles an N-terminally extended form of Xenopus neuromedin B. Ranatensin-HL and its C-terminal decapeptide (ranatensin-HL-10) were chemically synthesised and subjected to in vitro smooth muscle assays in which they were found to display moderate stimulatory effects on rat urinary bladder and uterus smooth muscles with EC50 values in the range of 1–10 nM. The prepro-ranatensin-HL was highly homological to a bombesin-like peptide from Rana catesbeiana at both nucleotide and amino acid levels, which might provide a clue for the taxonomic classification of ranid frogs in the future.

Simulation of <i>In Vitro</i>-Like Electrical Activities in Urinary Bladder Smooth Muscle Cells

Urinary bladder smooth muscle (UBSM) generates spontaneous electrical activities due to stochastic nature of purinergic neurotransmitter release from the parasympathetic nerve. The stochastic nature of the purinergic neurotransmitter release was represented by a simplified ‘point-conductance’ model to mimic in vitro-like electrical activities in UBSM cell. The point-conductance was represented by the independent synaptic conductance described by the stochastic random-walk processes and injected into a single-compartment model of mouse UBSM cell. This model successfully evoked irregular spontaneous depolarizations (SDs) and spontaneous action potential (sAP) as the properties of in vitro-like electrical activities in UBSM cells. The model mimics the T- and L-type Ca2+ ion channel blocker by setting their respective conductance to zero. We also found that the point-conductance model modulates the sAP properties by adding background activity.

A computational model of intracellular calcium oscillations in urinary bladder smooth muscle cells.

This paper presents a computational model for calcium (Ca2+) oscillations in detrusor smooth muscle (DSM) cells. The proposed model simulates the temporal profile of oscillations by incorporating various cellular and subcellular components. The cellular components include calcium influx via membrane and the plasma membrane calcium ATPase (PMCA) pump. The subcellular components include ryanodine receptors (RyRs), inositol 1, 4, 5 trisphosphate receptors (IP3Rs) and the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. The use of both cellular and subcellular components provides a better estimation of the origin and factors affecting these oscillations. Moreover, our work correlates these computational findings with associated physiology of the smooth muscle cell that aids our understanding of intracellular calcium oscillations and its inception in DSM. A deeper insight into calcium signalling in DSM cells is expected to provide a firmer basis for understanding the mechanical contractile activity.

Do β3-adrenoceptor agonists cause urinary bladder smooth muscle relaxation by inhibiting acetylcholine release?

The mechanisms leading to improvement of overactive bladder syndrome upon treatment with a β3-adrenoceptor agonist remain controversial. New research points to a role of inhibition of neuronal acetylcholine release. Whether this occurs by a direct effect on prejunctional β3-adrenoceptors or indirectly by formation of adenosine and activation of A1 adenosine receptors is being discussed.

Effect of distigmine on the contractile response of guinea pig urinary bladder to electrical field stimulation

Distigmine bromide (distigmine) is a reversible carbamate group cholinesterase (ChE) inhibitor. Although mainly used clinically for the treatment of myasthenia gravis, distigmine is also indicated for detrusor underactivity in Japan. According to the pharmacological classification of distigmine, its therapeutic effect against detrusor underactivity appears to be produced by enhanced urinary bladder smooth muscle (UBSM) contractility due to an increased concentration of acetylcholine between parasympathetic nerve endings and UBSM cells. However, ATP as well as acetylcholine is also released from parasympathetic nerve endings that dominate UBSM. The present study was thus carried out to investigate the potentiating effects of distigmine on the two UBSM contractile components in response to parasympathetic nerve stimulation induced by electrical field stimulation (EFS). In isolated guinea pig UBSM tissues, EFS (1–16Hz) produced tetrodotoxin-sensitive, frequency-dependent contractions. The contractile responses to EFS were largely diminished by atropine (10−6M), and the remaining contractile components in the presence of atropine were virtually abolished by α,β-methylene adenosine triphosphate (α,β-mATP) (10−4M). Distigmine (10−6M) significantly potentiated EFS-induced contractile components generated in the presence of α,β-mATP (10−4M), but did not potentiate EFS-induced contractile components generated in the presence of atropine (10−6M). These findings clearly indicate that distigmine strongly potentiates UBSM contraction selectively induced by parasympathetic nerve-derived acetylcholine, suggesting a potential mechanism by which distigmine restores detrusor underactivity.

A computational model of the [Formula: see text] transients and influence of buffering in guinea pig urinary bladder smooth muscle cells.

Many cellular events including electrical activity and muscle contraction are regulated and coordinated by intracellular [Formula: see text] concentration ([[Formula: see text]][Formula: see text]. In detrusor smooth muscle (DSM) cells, [[Formula: see text]]i is normally maintained at very low levels and rises transiently during signalling processes as a result of (i) influx from the extracellular space (mainly via L-type and T-type [Formula: see text] channels) and (ii) [Formula: see text] release from sarcoplasmic reticulum (SR) into the cytoplasm. Intracellular [Formula: see text] buffers, both fixed and diffusible, play a vital role in shaping the radial distribution of free [Formula: see text]. Our aim, in the work presented here, is to develop a mathematical model of [Formula: see text] buffering and diffusion and to generate [Formula: see text] transient in guinea pig DSM cells. The [Formula: see text] transient is generated using inward [Formula: see text] current that arises following voltage clamp and mediated by L-type and T-type [Formula: see text] channels. [Formula: see text] transient is obtained for different radial locations (or shells) of the DSM cytosol. This modeling study explores the levels of [[Formula: see text]]i achieved near the plasma membrane and in deeper locations. The [Formula: see text] transient generated in our model shows a high degree of similarity with experimental findings in terms of amplitude, duration and half-decay time. A number of different buffer properties such as concentration and mobility are tested for their effect on amplitude and shape of [Formula: see text] transient. The presence of fast buffer concentration in the cytosol markedly delays the rise of [[Formula: see text]]i in the core of the cell. Increase in the mobility of fast buffer slightly speeds up the redistribution of [Formula: see text]. To explore the model further, the role of plasma membrane [Formula: see text]-ATPase (PMCA) pump, sarcoplasmic/endoplasmic reticulum [Formula: see text]-ATPase (SERCA) pump and sodium calcium exchanger (NCX) on [Formula: see text] transient is studied and it is suggested that NCX may be of primary importance for the immediate lowering of [[Formula: see text]]i during the falling phase of a [Formula: see text] transient in DSM cells.

Expression and function of the small-conductance Ca2+-activated K+ channel is decreased in urinary bladder smooth muscle cells from female guinea pig with partial bladder outlet obstruction.

Overactive bladder (OAB), usually accompanied by partial bladder outlet obstruction (PBOO), is associated with detrusor overactivity (DO) which is related to the increased urinary bladder smooth muscle (UBSM) cells excitability. Small-conductance Ca2+-activated K+ (SK) channels play a constitutive regulatory role of UBSM excitability and contractility. PBOO is associated with the decreased SK channels mRNA expression and the attenuated regulative effect of SK channels on UBSM contractility. However, the regulation of SK channels in PBOO UBSM cell excitability is less clear. Here, we tested the hypothesis that PBOO is associated with decreased expression and function of SK channels in UBSM cells and that SK channels are a potential target for the treatment of OAB. Cystometry indicated that DO was achieved 2 weeks after PBOO in female guinea pigs. Using this animal model, we conducted single-cell quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and patch-clamp electrophysiology. The single-cell qRT-PCR experiments indicated the reduced SK channel mRNA expression in PBOO UBSM cells. Patch-clamp studies revealed that NS309 had a diminished effect on resting membrane potential hyperpolarization via the activation of SK channels in PBOO UBSM cells. Moreover, attenuated whole-cell SK channel currents were demonstrated in PBOO UBSM cells. The attenuated expression and function of SK channels, which results in the increased UBSM cells excitability and contributes to DO, was discovered in PBOO UBSM cells, suggesting that SK channels might be potential therapeutic targets for the control of OAB.

Three-dimensional mechano-electrochemical model for smooth muscle contraction of the urinary bladder

The urinary bladder is a central organ of vertebrates and imposes, based on its extreme deformation (volume changes up to several 100%), special requirements on the overall bladder tissue. However, studies focusing on three-dimensional modelling of bladder deformation and bladder function during micturition are rare. Based on three fields, namely, the membrane potential, calcium concentration, and placement, a mechano-electrochemical-coupled, three-dimensional model describing the contractile behaviour of urinary bladder smooth muscle is presented using a strain energy function. The strain energy functions for the different layers of the bladder wall are additively decomposed into a passive part comprising elastin, the extracellular matrix (ECM), and collagen and an active electrochemical-driven part comprising the contraction of smooth muscle cells (SMC). While the two-variable FitzHugh-Nagumo-type membrane model (FitzHugh, 1961; Nagumo et al., 1962) has been used to describe the membrane potential characteristics, the four-state, cross-bridge model of Hai and Murphy (1988) is implemented into the finite element method for the quantification of the calcium phase. Appropriate model parameters were determined experimentally using 40 tissue strips isolated from porcine bladders. Characteristic orientation-dependent passive and active stress-stretch relationships were identified for muscle strips, including the entire bladder wall structure and those featuring the isolated muscle layer only. Active experiments on the smooth muscle layers revealed higher stresses in the longitudinal (28.9kPa) direction than in the transversal (22.7kPa) one. Additionally, three-dimensional deformation characteristics were recorded from single muscle strips to qualitatively confirm the strip simulations. Three-dimensional simulations at the tissue strip level and the organ level were performed to analyse the interaction among the electrical action potential, calcium distribution, chemical degree of activation, and equivalent von Mises stress.

Discovery of Novel Indazole Derivatives as Orally Available β3-Adrenergic Receptor Agonists Lacking Off-Target-Based Cardiovascular Side Effects.

We previously discovered that indazole derivative 8 was a highly selective β3-adrenergic receptor (β3-AR) agonist, but it appeared to be metabolically unstable. To improve metabolic stability, further optimization of this scaffold was carried out. We focused on the sulfonamide moiety of this scaffold, which resulted in the discovery of compound 15 as a highly potent β3-AR agonist (EC50 = 18 nM) being inactive to β1-, β2-, and α1A-AR (β1/β3, β2/β3, and α1A/β3 > 556-fold). Compound 15 showed dose-dependent β3-AR-mediated responses in marmoset urinary bladder smooth muscle, had a desirable metabolic stability and pharmacokinetic profile (Cmax and AUC), and did not obviously affect heart rate or mean blood pressure when administered intravenously (3 mg/kg) to anesthetized rats. Thus, compound 15 is a highly potent, selective, and orally available β3-AR agonist, which may serve as a candidate drug for the treatment of overactive bladder without off-target-based cardiovascular side effects.

Cavin‐3 deficiency reduces caveolae abundance in vascular and urinary bladder smooth muscle cells

We investigated the effect of cavin‐3 (protein kinase C delta binding protein, Prkcdbp) deficiency on vascular and urinary bladder function. Small mesenteric arteries and the urinary bladder were isolated from mice lacking cavin‐3 (Prkcdbp−/−, knockout: KO) and from control littermates (Prkcdbp+/+, wild type: WT). One group of mice was used for blood pressure measurements, whereas another set of mice underwent bladder outlet obstruction followed by analysis of protein expression using western blotting. Our studies revealed that loss of cavin‐3 resulted in a 40% reduction of the caveola protein cavin‐1 (polymerase I and transcript release factor, Ptrf) in vascular and bladder smooth muscle. Electron microscopy demonstrated that loss of cavin‐3 was accompanied by a reduction of caveolae by 40% in smooth muscle, while the abundance of caveolae in endothelial cells was unchanged. Vascular contraction in response to cirazoline (α1‐adrenergic agonist) was normal, but nitric‐oxide dependent relaxation was enhanced. The latter effect was associated with elevated expression of soluble guanylyl cyclase, and yet blood pressure was similar in WT and KO mice. Contraction of the urinary bladder was not affected by loss of cavin‐3, and the proteomic response to outlet obstruction, including STAT3 (signal transducer and activator of transcription 3) phosphorylation, induction of synthetic markers, and repression of contractile markers were identical, the only exception being a curtailed induction of the Golgi protein GM130 (Golgi matrix protein 130 kD). In conclusion, loss of cavin‐3 thus reduces the number of caveolae in smooth muscle and partly destabilizes cavin‐1, but the functional consequences are modest and include an elevated vascular sensitivity to nitric oxide and a somewhat disturbed Golgi homeostasis in a situation of severe cellular stress.Support or Funding InformationThis study was funded by The Swedish Research Council (Vetenskapsrådet, http://www.vr.se/, K2015‐65X‐22662‐01‐3/KS), the Crafoord Foundation (http://en.crafoord.se/, CR), and the Royal Physiographic Society (Kungliga Fysiografiska Sällskapet, http://www.fysiografen.se/, CR).