Urinary Bladder Smooth Muscle Excitability Research Articles

Compound 48/80 increases murine bladder wall compliance independent of mast cells

A balance between stiffness and compliance is essential to normal bladder function, and changes in the mechanical properties of the bladder wall occur in many bladder pathologies. These changes are often associated with the release of basic secretagogues that in turn drive the release of inflammatory mediators from mast cells. Mast cell degranulation by basic secretagogues is thought to occur by activating an orphan receptor, Mas-related G protein-coupled receptor B2 (Mrgprb2). We explored the effects of the putative mast cell degranulator and Mrgprb2 agonist Compound 48/80 on urinary bladder wall mechanical compliance, smooth muscle contractility, and urodynamics, and if these effects were mast cell dependent. In wild-type mice, Mrgprb2 receptor mRNA was expressed in both the urothelium and smooth muscle layers. Intravesical instillation of Compound 48/80 decreased intermicturition interval and void volume, indicative of bladder overactivity. Compound 48/80 also increased bladder compliance while simultaneously increasing the amplitude and leading slope of transient pressure events during ex vivo filling and these effects were inhibited by the Mrgprb2 antagonist QWF. Surprisingly, all effects of Compound 48/80 persisted in mast cell-deficient mice, suggesting these effects were independent of mast cells. These findings suggest that Compound 48/80 degrades extracellular matrix and increases urinary bladder smooth muscle excitability through activation of Mrgprb2 receptors located outside of mast cells. Thus, the pharmacology and physiology of Mrgprb2 in the urinary bladder is of potential interest and importance in terms of treating lower urinary tract dysfunction.

Differential effects of TRPM4 channel inhibitors on Guinea pig urinary bladder smooth muscle excitability and contractility: Novel 4-chloro-2-[2-(2-chloro-phenoxy)-acetylamino]-benzoic acid (CBA) versus classical 9-phenanthrol.

Non‐selective cation channels in urinary bladder smooth muscle (UBSM) are thought to mediate increases in cellular excitability and contractility. For transient receptor potential melastatin type‐4 (TRPM4) channels, the evidence primarily relies on the inhibitor 9‐phenanthrol, which exhibits pharmacological limitations. Recently, 4‐chloro‐2‐[2‐(2‐chloro‐phenoxy)‐acetylamino]‐benzoic acid (CBA) has been discovered as a novel TRPM4 channel blocker. We examined how, in comparison to 9‐phenanthrol, CBA affects the excitability of freshly isolated guinea pig UBSM cells and the contractility of UBSM strips. Additionally, non‐selective TRPM4 channel inhibitor flufenamic acid (FFA) and potentiator BTP2 (also known as YM‐58483) were studied in UBSM cells. Unlike robust inhibition for 9‐phenanthrol already known, CBA (up to 100 μM) displayed either no or a very weak reduction (<20%) in spontaneous phasic, 20 mM KCl‐induced, and electrical field stimulated contractions. For 300 μM CBA, reductions were higher except for an increase in the frequency of KCl‐induced contractions. In UBSM cells, examined under amphotericin B‐perforated patch‐clamp, CBA (30 μM) did not affect the membrane potential (I = 0) or voltage step‐induced whole‐cell cation currents, sensitive to 9‐phenanthrol. The currents were not inhibited by FFA (100 μM), increased by BTP2 (10 μM), nor enhanced under a strongly depolarizing holding voltage of −16 or + 6 mV (vs. −74 mV). None of the three compounds affected the cell capacitance, unlike 9‐phenanthrol. In summary, the novel inhibitor CBA and nonselective FFA did not mimic the inhibitory properties of 9‐phenanthrol on UBSM function. These results suggest that TRPM4 channels, although expressed in UBSM, play a distinct role rather than direct regulation of excitability and contractility.

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.

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.

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.

Testosterone decreases urinary bladder smooth muscle excitability via novel signaling mechanism involving direct activation of the BK channels.

In addition to improving sexual function, testosterone has been reported to have beneficial effects in ameliorating lower urinary tract symptoms by increasing bladder capacity and compliance, while decreasing bladder pressure. However, the cellular mechanisms by which testosterone regulates detrusor smooth muscle (DSM) excitability have not been elucidated. Here, we used amphotericin-B perforated whole cell patch-clamp and single channel recordings on inside-out excised membrane patches to investigate the regulatory role of testosterone in guinea pig DSM excitability. Testosterone (100 nM) significantly increased the depolarization-induced whole cell outward currents in DSM cells. The selective pharmacological inhibition of the large-conductance voltage- and Ca2+-activated K+ (BK) channels with paxilline (1 μM) completely abolished this stimulatory effect of testosterone, suggesting a mechanism involving BK channels. At a holding potential of -20 mV, DSM cells exhibited transient BK currents (TBKCs). Testosterone (100 nM) significantly increased TBKC activity in DSM cells. In current-clamp mode, testosterone (100 nM) significantly hyperpolarized the DSM cell resting membrane potential and increased spontaneous transient hyperpolarizations. Testosterone (100 nM) rapidly increased the single BK channel open probability in inside-out excised membrane patches from DSM cells, clearly suggesting a direct BK channel activation via a nongenomic mechanism. Live-cell Ca2+ imaging showed that testosterone (100 nM) caused a decrease in global intracellular Ca2+ concentration, consistent with testosterone-induced membrane hyperpolarization. In conclusion, the data provide compelling mechanistic evidence that under physiological conditions, testosterone at nanomolar concentrations directly activates BK channels in DSM cells, independent from genomic testosterone receptors, and thus regulates DSM excitability.

Pharmacological Opening of KV7 Channels by the Novel Activator ML‐213: Role in Guinea Pig Urinary Bladder Smooth Muscle Function

It has been recently suggested that voltage‐gated KV7 channels (KV7.1‐KV7.5) regulate urinary bladder smooth muscle (UBSM) function. Despite emerging developments, the physiological role of individual KV7 channel subtypes remains less clear. Here, we utilized the novel compound N‐(2,4,6‐Trimethylphenyl)‐bicyclo[2.2.1]heptane‐2‐carboxamide (ML‐213), a potent activator of KV7.2, KV7.4, and KV7.5 channels, to elucidate their physiological roles in guinea pig UBSM function. Using isometric UBSM tension recordings, Ca2+ imaging, and amphotericin‐B perforated patch‐clamp electrophysiology, we elucidated the role of ML‐213‐sensitive KV7 channels in regulating UBSM excitability and contractility. In functional studies of UBSM contractility, ML‐213 concentration‐dependently (100 nM‐30 μM) inhibited spontaneous phasic, pharmacologically‐induced, and nerve‐evoked contractions in UBSM isolated strips. In UBSM strips loaded with the ratiometric fluorescence probe fura 2, ML‐213 (10 μM) decreased the global intracellular Ca2+ concentration and inhibited spontaneous Ca2+ transients, which is consistent with the inhibitory effects on UBSM contractility. ML‐213‐induced attenuation of global Ca2+ levels was abolished in the presence of the L‐type voltage‐gated Ca2+ channel inhibitor nifedipine (1 μM) and the KV7.1–KV7.5 channel inhibitor XE991 (10 μM). These data suggests that ML‐213 decreases the global intracellular Ca2+ concentration by inhibiting L‐type voltage‐gated Ca2+ channels through an indirect mechanism downstream from KV7 channel activation. In current‐clamp mode of the perforated patch‐clamp technique, ML‐213 hyperpolarized the cell membrane potential and inhibited spontaneous action potentials in UBSM cells. ML‐213‐induced hyperpolarization of the UBSM cell membrane potential was reversible by washout of the compound. We next aimed to examine the effects of ML‐213 on voltage‐step depolarization‐induced whole cell KV7 currents using the perforated patch‐clamp technique in voltage‐clamp mode. To isolate KV7 currents, the extracellular bath solution contained the large conductance voltage‐ and Ca2+‐activated K+ channel inhibitor paxilline (1 μM) and gadolinium chloride (GdCl3, 100 μM), which blocks L‐type voltage‐gated Ca2+ channels and non‐selective cation channels. Under these experimental conditions, ML‐213 (10 μM) enhanced whole cell KV7 currents. These findings suggest that the modulation of K+ transport through ML‐213‐sensitive KV7 channels underlies ML‐213‐induced cell membrane hyperpolarization to decrease the global intracellular Ca2+ concentration and UBSM contractility. These combined results, using the novel compound ML‐213, suggest that KV7.2‐, KV7.4‐, and KV7.5‐containing channels are essential regulators of the excitability and contractility of UBSM by virtue of their control of the resting membrane potential. In addition, these studies provide a foundational basis for further studies investigating KV7 channel functional roles in human UBSM excitability and contractility to confirm their potential as novel therapeutic targets for bladder dysfunction.Support or Funding InformationSupported by NIH grant R01‐DK106964 to Georgi V. Petkov and F31‐DK104528 to Aaron Provence.

Phosphodiesterase Type-1 Regulates Transient BK Currents and Contractility of Human Urinary Bladder Smooth Muscle

Large conductance Ca2+-activated K+ (BK) channels are critical regulators of urinary bladder smooth muscle (UBSM) function. We aimed to investigate phosphodiesterase type 1 (PDE1) interactions with BK channels in human UBSM to determine the mechanism by which PDE1 regulates human urinary bladder function. A combined electrophysiological, functional, and pharmacological approach was applied using human UBSM specimens obtained from open bladder surgeries. The perforated whole cell patch-clamp technique was used to record transient BK currents (TBKCs) and the cell membrane potential in freshly-isolated human UBSM cells in combination with the selective PDE1 inhibitor, 8-methoxymethyl-3-isobutyl-1-methylxanthine (8MM-IBMX). Isometric tension recordings were used to measure spontaneous phasic and electrical field stimulation (EFS)-induced contractions in human UBSM isolated strips. Selective pharmacological inhibition of PDE1 with 8MM-IBMX (10 μM) increased TBKC activity in human UBSM cells, which was abolished by subsequent inhibition of protein kinase A (PKA) with H-89 (10 μM). The stimulatory effect of 8MM-IBMX on TBKCs was reversed upon activation of muscarinic acetylcholine receptors with carbachol (1 μM). 8MM-IBMX (10 μM) hyperpolarized the UBSM cell membrane potential, an effect blocked by PKA inhibition. 8MM-IBMX significantly decreased spontaneous phasic and nerve-evoked contractions of human UBSM isolated strips.The results reveal that pharmacological inhibition of PDE1 attenuates human UBSM excitability and contractility by activating BK channels via a PKA-dependent mechanism. The data also suggest interactions between PDE1 and muscarinic signaling pathways in human UBSM. Overall, our data provide mechanistic evidence that pharmacological inhibition of PDE1 can be a useful therapeutic approach for the treatment of overactive bladder syndrome by attenuating UBSM excitability and contractility. These investigations are an important step in validating PDE1 as a potential novel therapeutic target for overactive bladder.Supported by NIH R01DK106964 and R01DK084284 to Georgi V. Petkov.

The role of large conductance voltage‐ and Ca 2+ ‐activated K + channels as low affinity receptors for 17β‐estradiol in guinea pig urinary bladder smooth muscle excitability

The role of large conductance voltage‐ and Ca ²⁺ ‐activated K ⁺ channels as low affinity receptors for 17β‐estradiol in guinea pig urinary bladder smooth muscle excitability

Functional roles for K V 7.2 and K V 7.3 channels in guinea pig urinary bladder smooth muscle revealed by the novel K V 7.2/K V 7.3 channel opener ICA‐069673

We elucidated the physiological role of KV7.2/KV7.3 channels in guinea pig urinary bladder smooth muscle (UBSM) in vitro using the novel KV7.2/KV7.3 channel opener ICA-069673 and a multidisciplinary experimental approach. Western blot revealed protein expression for KV7.2 and KV7.3 channels in UBSM. In isolated UBSM cells, immunocytochemistry detected protein expression of KV7.2 and KV7.3 channels with localization at the cell membrane. ICA-069673 caused a concentration-dependent (100 nM-30 µM) inhibition of spontaneous phasic, pharmacologically-induced, and nerve-evoked contractions in UBSM isolated strips. In solution with elevated K+ concentration (60 mM), the inhibitory effects of ICA-069673 on UBSM contractility were attenuated. ICA-069673 decreased the global intracellular Ca2+ concentration in UBSM cells, an effect blocked by the L-type voltage-gated Ca2+ channel inhibitor nifedipine (1 µM). ICA-069673 significantly hyperpolarized the membrane potential and inhibited spontaneous action potentials in UBSM cells measured with perforated patch-clamp. XE991 (10 µM), a Kv7 channel inhibitor, abolished the effects of ICA069673 on USBM contractility and membrane potential. These results establish KV7.2/KV7.3 channels as critical regulators of UBSM excitability and contractility. Supported by NIH R01DK04284 to Georgi V. Petkov & NIH F31DK104528 to Aaron Provence.

Large conductance voltage‐ and Ca 2+ ‐activated K + channels mediate the 17β‐estradiol inhibitory effects on human urinary bladder smooth muscle cell excitability and contractility

Estrogens are critical regulators of smooth muscle function, including urinary bladder smooth muscle (UBSM) contractility. However, the mechanism underlying estrogen's regulatory role in UBSM has not been fully elucidated. Here, we explored whether 17β-estradiol,a physiologically active estrogen, activates large conductance voltage- and Ca2+-activated K+ (BK) channels in human UBSM. We obtained UBSM samples from 18 patients to examine the physiological role of 17β-estradiol in regulating UBSM function. 17β-estradiol inhibited UBSM spontaneous phasic contractions in a concentration-dependent manner (100 nM-1 µM). These 17β-estradiol inhibitory effects were prevented in the presence of paxilline (1 µM), a selective inhibitor of BK channels. 17β-estradiol (100 nM) also inhibited nerved-evoked contractions of UBSM isolated strips. Perforated patch-clamp data showed that 17β-estradiol increased the depolarization-induced whole cell outward currents, effects that were blocked by paxilline (1 µM). 17β-estradiol (100 nM) also increased the frequency of the transient BK currents in human UBSM cells. 17β-estradiol (100 nM) increased the BK channel open probability in inside-out excised patches from UBSM cells, supporting the concept that 17β-estradiol activates BK channels directly. Our results suggest that 17β-estradiol has a direct stimulatory effect on BK channel activity, thus decreasing the human UBSM excitability and contractility. Supported by NIH R01DK04284 to G.V. Petkov & NIH F31DK104528 to A. Provence.

Evaluation of mouse urinary bladder smooth muscle for diurnal differences in contractile properties.

Most physiological systems show daily variations in functional output, entrained to the day–night cycle. Humans exhibit a daily rhythm in urinary voiding (micturition), and disruption of this rhythm (nocturia) has significant clinical impact. However, the underlying mechanisms are not well-understood. Recently, a circadian rhythm in micturition was demonstrated in rodents, correlated with functional changes in urodynamics, providing the opportunity to address this issue in an animal model. Smooth muscle cells from mouse bladder have been proposed to express a functional and autonomous circadian clock at the molecular level. In this study, we addressed whether a semi-intact preparation of mouse urinary bladder smooth muscle (UBSM) exhibited measurable differences in contractility between day and night. UBSM tissue strips were harvested at four time points over the diurnal cycle, and spontaneous (phasic) and nerve-evoked contractions were assessed using isometric tension recordings. During the active period (ZT12-24) when micturition frequency is higher in rodents, UBSM strips had no significant differences in maximal- (high K+) or nerve-evoked contractions compared to strips harvested from the resting period (ZT0-12). However, a diurnal rhythm in phasic contraction was observed, with higher amplitudes at ZT10. Consistent with the enhanced phasic amplitudes, expression of the BK K+ channel, a key suppressor of UBSM excitability, was lower at ZT8. Higher expression of BK at ZT20 was correlated with an enhanced effect of the BK antagonist paxilline (PAX) on phasic amplitude, but PAX had no significant time-of-day dependent effect on phasic frequency or nerve-evoked contractions. Overall, these results identify a diurnal difference for one contractile parameter of bladder muscle. Taken together, the results suggest that autonomous clocks in UBSM make only a limited contribution to the integrated control of diurnal micturition patterns.

Central role of the BK channel in urinary bladder smooth muscle physiology and pathophysiology.

The physiological functions of the urinary bladder are to store and periodically expel urine. These tasks are facilitated by the contraction and relaxation of the urinary bladder smooth muscle (UBSM), also known as detrusor smooth muscle, which comprises the bladder wall. The large-conductance voltage- and Ca(2+)-activated K(+) (BK, BKCa, MaxiK, Slo1, or KCa1.1) channel is highly expressed in UBSM and is arguably the most important physiologically relevant K(+) channel that regulates UBSM function. Its significance arises from the fact that the BK channel is the only K(+) channel that is activated by increases in both voltage and intracellular Ca(2+). The BK channels control UBSM excitability and contractility by maintaining the resting membrane potential and shaping the repolarization phase of the spontaneous action potentials that determine UBSM spontaneous rhythmic contractility. In UBSM, these channels have complex regulatory mechanisms involving integrated intracellular Ca(2+) signals, protein kinases, phosphodiesterases, and close functional interactions with muscarinic and β-adrenergic receptors. BK channel dysfunction is implicated in some forms of bladder pathologies, such as detrusor overactivity, and related overactive bladder. This review article summarizes the current state of knowledge of the functional role of UBSM BK channels under normal and pathophysiological conditions and provides new insight toward the BK channels as targets for pharmacological or genetic control of UBSM function. Modulation of UBSM BK channels can occur by directly or indirectly targeting their regulatory mechanisms, which has the potential to provide novel therapeutic approaches for bladder dysfunction, such as overactive bladder and detrusor underactivity.

Role of phosphodiesterase‐1 in muscarinic receptor‐induced human urinary bladder smooth muscle excitability and contractility (865.4)

Role of phosphodiesterase‐1 in muscarinic receptor‐induced human urinary bladder smooth muscle excitability and contractility Wenkuan Xin1, Ning Li1, Eric S. Rovner2, Qiuping Cheng1, and Georgi V. Petkov1,2 1Drug Discovery &amp; Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208 and 2Medical University of South Carolina, Charleston, SC 29425 Activation of muscarinic M3 receptors (M3Rs) leads to an inhibition of the large conductance Ca2+‐activated K+ (BK) channels and an increase in human urinary bladder smooth muscle (UBSM) excitability. M3R‐mediated Ca2+ influx activates phosphodiesterase‐1 (PDE1) and reduces cellular cAMP levels. We investigated the mechanism by which the integrated PDE1 and MR signaling pathways stimulate human UBSM excitability and contractility. Pharmacological activation of M3Rs with carbachol (1 µM) decreased the frequency and amplitude of transient BK currents (TBKCs) in UBSM cells and subsequent inhibition of PDE1 with 8MM‐IBMX (10 µM) recovered TBKC activity. In the absence of M3R stimulation, inhibition of PDE1 increased TBKCs frequency, and subsequent inhibition of protein kinase A (PKA) with H‐89 (10 µM) reduced TBKCs frequency. 8MM‐IBMX (10 µM) hyperpolarized UBSM membrane potential, an effect which was blocked upon PKA inhibition. 8MM‐IBMX suppressed spontaneous phasic contractions and nerve‐evoked contractions of UBSM isolated strips. In conclusion, PDE1 enhances the M3R‐mediated inhibition of BK channels. Inhibition of PDE1 attenuates UBSM excitability by increasing BK channel activity via a PKA‐dependent mechanism. A combination of a PDE1 inhibitor and a MR antagonist can be an effective novel approach for the treatment of bladder overactivity.Key words: phosphodiesterase, muscarinic receptor, detrusorGrant Funding Source: NIH R01 DK084284 to Georgi V. Petkov

The large conductance Ca2+‐activated K+ channel: a key intermediate in cholinergic regulation of human urinary bladder smooth muscle excitability (865.5)

We investigated whether muscarinic receptor (MR) activation increases human urinary bladder smooth muscle (UBSM) excitability by directly or indirectly inhibiting the large conductance Ca2+‐activated K+ (BK) channels. We used the MR agonist, carbachol, to determine the changes in BK channel activity in freshly‐isolated human UBSM cells using the perforated whole cell and single BK channel patch‐clamp recordings. Carbachol inhibited the amplitude and frequency of BK channel‐mediated spontaneous transient outward currents and spontaneous transient hyperpolarizations, both triggered by ryanodine receptor‐dependent Ca2+ release. Carbachol also caused UBSM cell depolarization, which was not observed in the presence of iberiotoxin, a BK channel inhibitor. The potential direct effects of carbachol on BK channels were examined under conditions of removing all major cellular Ca2+ sources for BK channel activation with pharmacological inhibitors (thapsigargin, ryanodine, and nifedipine). In the presence of these inhibitors, carbachol did not affect the single BK channel open probability and mean BK channel conductance (cell‐attached configuration) or the whole cell BK currents. The data support the concept that MR activation triggers indirect BK channel inhibition mediated by intracellular Ca2+, thus increasing human UBSM cell excitability.Grant Funding Source: Supported by NIH R01 DK084284 to Georgi V. Petkov.

Control of urinary bladder smooth muscle excitability by the TRPM4 channel modulator 9-phenanthrol

The Ca2+-activated monovalent cation selective transient receptor potential melastatin 4 (TRPM4) channel has been recently identified in detrusor smooth muscle (DSM) of the urinary bladder. Two recent publications by our research group provide evidence in support of the novel hypothesis that TRPM4 channels enhance DSM excitability and contractility. This is a critical question as prior studies have primarily targeted hyperpolarizing currents facilitated by K+ channels, but the depolarizing component in DSM cells is not well understood. For the first time, we utilized the selective TRPM4 channel inhibitor, 9-phenanthrol, to investigate TRPM4 channel functional effects in DSM at both cellular and tissue levels in rodents. Our new data presented here showed that in rat DSM cells, 9-phenanthrol attenuates spontaneous inward currents in the presence of the muscarinic receptor agonist, carbachol, thus reducing DSM cell excitability. In support of our original hypothesis, we found that TRPM4 channel mRNA levels are much higher in DSM vs. vascular smooth muscle and that inhibition of TRPM4 channels can potentially attenuate DSM excitability. Thus, we postulate the novel concept that selective pharmacological inhibition of TRPM4 channels can limit both excitability and contractility of DSM.

Constitutively active phosphodiesterase type 4 controls large conductance Ca2+‐activated K+ channel activity in guinea pig urinary bladder smooth muscle

Nonselective pharmacological inhibition of phosphodiesterases (PDE) elevates cellular cAMP levels, leading to activation of the large conductance Ca2+‐activated K+ (BK) channels and attenuation of guinea pig urinary bladder smooth muscle (UBSM) excitability and contractility. Here, we investigated the mechanism of selective inhibition of cAMP‐specific PDE4 on UBSM excitability and contractility using the perforated patch‐clamp technique on isolated UBSM cells and isometric tension recordings of UBSM isolated strips. Our data showed that selective PDE4‐inhibition with rolipram increased the spontaneous transient BK currents frequency and hyperpolarized the cell resting membrane potential (RMP). Blocking BK channels with paxilline reversed the hyperpolarization effect of rolipram and depolarized the RMP back to the control levels. PDE4‐inhibition did not cause RMP hyperpolarization in the presence of cAMP‐dependent protein kinase (PKA) inhibitor, H‐89. PDE4 inhibition reduced the UBSM spontaneous phasic and carbachol‐induced contraction frequency and amplitude, as well as the electrical‐field‐stimulation‐induced contraction amplitude. In conclusion, selective inhibition of the constitutively active PDE4 suppressed the guinea pig UBSM excitability and contractility via a BK channel‐dependent mechanism involving the PKA signaling pathway. Supported by NIH DK08424 to GVP.

Diurnal Variation in Mouse Urinary Bladder Smooth Muscle (UBSM) Contractility

Physiological output varies by time of day, coordinated by the central circadian clock in the hypothalamus. Recently, a circadian rhythm in micturition was demonstrated in rodents, correlated with a day‐night change in the storage and voiding of the bladder. The goal of this study was to determine whether isolated UBSM exhibited an intrinsic difference in contractility between day and night. Mouse UBSM strips were harvested at 4 time points over the circadian cycle, zeitgeber time (ZT) 2, 8, 14, and 20, and phasic and nerve‐evoked (EFS) contractions were investigated using isometric tension recordings. We hypothesized that UBSM harvested from mice during the active period when micturition peaks (ZT8–14) would demonstrate stronger contractile activity than strips harvested during the rest period (ZT20–2), when the bladder relaxes to store urine. Compared to ZT20, at ZT8 UBSM strips exhibited larger KCl‐induced (10 ± 0.4 versus 7 ± 0.4 mN), phasic (0.08 ± 0.02 versus 0.03 ± 0.01 mN), and EFS‐induced contractions (28 ± 3 versus 22 ± 2 mN, n = 6 mice per condition). Increased contractile amplitudes at ZT8 were correlated with lower expression of the BK K+ channel, a regulator of UBSM excitability. These results identify a diurnal difference in UBSM contractility in the absence of central control, and suggest that daily regulation of bladder excitability occurs locally within smooth muscle tissue. Funded by NIDDK R21089337.

Effect of GsMTx4 on Mouse Urinary Bladder Smooth Muscle (UBSM) Contractility

Urinary incontinence is a common problem with limited treatment options. Stretch‐activated channels (SACs) enhance UBSM excitability, and have been proposed as a novel target for reducing overactive bladder. The goal of this study was to determine whether an inhibitor of SACs (GsMTx4) decreased UBSM contractions in wild‐type (WT) and BK KO (Kcnma1−/−) bladders, a mouse model for urinary incontinence which harbors a deletion of the BK K+ channel. GsMTx4 was applied to isolated UBSM strips at 0.1–10 μM and spontaneous (phasic) and nerve‐evoked (EFS) contractions were investigated using isometric tension recordings. As expected, baseline BK KO contractile amplitudes were significantly larger than WT. Application of 5 μM GsMTx4 induced a smaller reduction in BK KO phasic amplitude than in WT (KO: −12 ± 0.22%, WT: −31 ± 0.04%), and reliably decreased phasic frequency in BK KO strips (KO: −14 ± 9%, WT: +11 ± 4%). Additionally, 10 μM GsMTx4 suppressed EFS evoked contractions (10 Hz amplitude, BK KO: −19 ± 12%, WT: −4 ± 1%). This data suggests that GsMTx4 may be effective at reducing spontaneous and nerve evoked contractions in overactive UBSM, while having a limited effect on normal UBSM. This work was funded by NIDDK R21–089337 (A.L.M.) and the APS UGSRF (Z.K.).

Constitutively active phosphodiesterase activity regulates urinary bladder smooth muscle function: critical role of KCa1.1 channel

Pharmacological blockade of cyclic nucleotide phosphodiesterase (PDE) can relax human urinary bladder smooth muscle (UBSM); however, the underlying cellular mechanism is unknown. In this study, we investigated the effects of PDE pharmacological blockade on human UBSM excitability, spontaneous and nerve-evoked contractility, and determined the underlying cellular mechanism mediating these effects. Patch-clamp electrophysiological experiments showed that 3-isobutyl-1-methylxanthine (10 μM), a nonselective PDE inhibitor, caused ∼3.6-fold increase in the transient K(Ca)1.1 channel current frequency and ∼2.5-fold increase in the spontaneous transient hyperpolarization frequency in UBSM-isolated cells. PDE blockade also caused ∼5.6-mV hyperpolarization of the UBSM cell membrane potential. Blocking the K(Ca)1.1 channels with paxilline abolished the spontaneous transient hyperpolarization and the hyperpolarization effect of PDE blockade on the UBSM cell membrane potential. Live cell Ca(2+)-imaging experiments showed that PDE blockade significantly decreased the global intracellular Ca(2+) levels. Attenuation of PDE activity significantly reduced spontaneous phasic contraction amplitude, muscle force integral, duration, frequency, and muscle tone of human UBSM isolated strips. Blockade of PDE also significantly reduced the contraction amplitude, muscle force integral, and duration of the nerve-evoked contractions induced by 20-Hz electrical field stimulation. Pharmacological inhibition of K(Ca)1.1 channels abolished the relaxation effects of PDE blockade on both spontaneous and nerve-evoked contractions in human UBSM-isolated strips. Our data provide strong evidence that in human UBSM PDE is constitutively active, thus maintaining spontaneous UBSM contractility. PDE blockade causes relaxation of human UBSM by increasing transient K(Ca)1.1 channel current activity, hyperpolarizing cell membrane potential, and decreasing the global intracellular Ca(2+).