Urinary Bladder Smooth Muscle Contractility Research Articles

The mast cell stimulator compound 48/80 causes urothelium-dependent increases in murine urinary bladder contractility.

Mast cells and degranulation of preformed inflammatory mediators contribute to lower urinary tract symptoms. This study investigated pathways by which the mast cell stimulator compound 48/80 alters urinary bladder smooth muscle contractility via mast cell activation. We hypothesized that 1) mast cell degranulation causes spontaneous urinary bladder smooth muscle contractions and 2) these contractions are caused by urothelium-derived PGE2. Urothelium-intact and -denuded urinary bladder strips were collected from mast cell-sufficient (C57Bl/6) and mast cell-deficient (B6.Cg-Kitw-sh) mice to determine if compound 48/80 altered urinary bladder smooth muscle (UBSM) contractility. Electrical field stimulation was used to assess the effects of compound 48/80 on nerve-evoked contractions. Antagonists/inhibitors were used to identify prostanoid signaling pathways activated or if direct activation of nerves was involved. Compound 48/80 caused slow-developing contractions, increased phasic activity, and augmented nerve-evoked responses in both mast cell-sufficient and -deficient mice. Nerve blockade had no effect on these responses; however, they were eliminated by removing the urothelium. Blockade of P2 purinoreceptors, cyclooxygenases, or G protein signaling abolished compound 48/80 responses. However, only combined blockade of PGE2 (EP1), PGF2α (FP), and thromboxane A2 (TP) receptors inhibited compound 48/80-induced responses. Thus, the effects of compound 48/80 are urothelium dependent but independent of mast cells. Furthermore, these effects are mediated by druggable inflammatory pathways that may be used to manage inflammatory nonneurogenic bladder hyperactivity. Finally, these data strongly suggest that great care must be taken when using compound 48/80 to determine mast cell-dependent responses in the urinary bladder.NEW & NOTEWORTHY Urothelial cells are first responders to noxious contents of the urine. Our study demonstrates that the urothelium is not only a barrier but also a modulator of urinary bladder smooth muscle phasic activity and contractility independent of immune cell recruitment in response to an inflammatory insult.

The urothelium drives changes to urinary bladder smooth muscle contractility that mimic neurogenic inflammatory signaling

Neurogenic inflammation disrupts urothelial homeostasis, which leads to barrier injury, cellular turnover, and dysregulated release of ATP and prostaglandins resulting in urinary bladder smooth muscle instability. Increased urothelium signaling is implicated in many bladder pathologies, such as idiopathic detrusor overactivity, interstitial cystitis, and recurrent urinary tract infections, in animal models along with patients. Previously, we utilized compound 48/80 as a pro-inflammatory agent on urinary bladder smooth muscle strips, which resulted in urothelium-dependent increases in smooth muscle phasic activity and augmented nerve-evoked contractions. Thus, we hypothesized that compound 48/80 causes spontaneous rhythmic contractions dependent on the release of urothelial-derived prostaglandins (PGE2, PGF2 alpha, Thromboxane A2) and pannexin-1-mediated ATP release, similar to neurogenic signaling pathways that drive smooth muscle hyperactivity. Tissue bath studies were performed with urothelium intact or denuded urinary bladder smooth muscle strips from 8–12-week-old male C57BL/6 mice (n=1 strip from N=5-6) to measure isometric contractility and nerve-evoked contractions in the presence of pharmacological inhibitors and compound 48/80 (10 μg/mL). Prior to compound 48/80 exposure, strips of urothelium were returned to the bottom of the organ tissue bath chambers directly underneath denuded smooth muscle strips to assess if soluble mediators were released. The non-selective cyclooxygenase inhibitor indomethacin (5 μM) diminished compound 48/80 augmentation of nerve-evoked contractions. However, the neurokinin 1 antagonist QWF (10 μM), PGF2 alpha FP receptor antagonist AL8810 (5 μM), PGE2 EP1 receptor antagonist SC51089 (10 μM), Thromboxane A2 TP receptor antagonist GR32191B (1 μM), and pannexin-1 mimetic inhibitory peptide 10Panx (200 μM) had no effect on compound 48/80-induced contractions compared to respective vehicle controls ( p=0.18, 0.91, 0.57, 0.41, and 0.44, respectively). A cocktail of all prostaglandin antagonists did significantly reduce contractions ( p=0.01). Interestingly, despite the physical distance of the detached urothelium, exposure to compound 48/80 resulted in increased contractility ( p=0.016) and phasic activity ( p=0.017) as compared to a urothelium-denuded strips. These results suggest that activation of urothelial signaling via compound 48/80 mimics neurogenic inflammation to release a wide array of prostaglandins capable of altering bladder smooth muscle contractility. Thus, the urothelium should be considered as an inflammatory milieu whereby physiological and pharmacological changes contribute to urinary bladder smooth muscle dysfunction. This research is supported by NIH P20-DK127554 (NRT), NIH R01-DK119615 (NRT & GCM; supplement to BMJ) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

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.

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.

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.

In Mice, KCNQ Channel Modulators Affect Afferent Nerve Activity and not Urinary Bladder Smooth Muscle Contractility Directly

KCNQ channels are a family of voltage‐dependent K+ channels, which open in response to membrane depolarization to regulate cell excitability. KCNQ activators, such as retigabine, have been used successfully to treat epilepsy; interestingly, these drugs also caused urinary retention. Using ex vivo measurements of bladder pressure and afferent nerve activity, isometric contractility of bladder strips, and electrophysiological recordings from freshly isolated urinary bladder smooth muscle (UBSM) cells, we explored the role of KCNQ channels as regulators of murine urinary bladder function. In the ex vivo bladder, both the KCNQ activator retigabine and the voltage‐gated Ca2+ channel inhibitor diltiazem blocked transient contractions (TCs) and associated bursts of afferent nerve activity. In addition, retigabine reduced baseline afferent nerve activity in the absence of TCs. Simulating TCs by mechanical deformation of the bladder wall also caused bursts of afferent activity; retigabine, but not diltiazem, inhibited these afferent bursts as well. This suggests that the effects of retigabine were not limited to detrusor muscle, and also included inhibition of afferent nerve activity. In bladder strips, neither retigabine (10 μM) nor the KCNQ antagonist XE‐991 (10 μM) altered contractions in response to electrical field stimulation (0.5 – 50 Hz). Retigabine did, however, significantly reduce 60 mM KCl‐induced contractions, possibly indicative of voltage‐dependent Ca2+ channel inhibition and not KCNQ activation. Consistent with contractility data, retigabine inhibited currents through voltage‐dependent Ca2+ channels and had no effect on K+ currents, as measured in UBSM cells using the patch‐clamp technique. Lastly, retigabine had no effect on resting membrane potential in UBSM cells, using the current‐clamp configuration of the patch clamp technique. Taken together, these data indicate that UBSM cells lack functional KCNQ channels, and the effects of retigabine on detrusor contractility may be due instead to non‐specific inhibition of voltage‐gated Ca2+ channels. Alternatively, urinary retention caused by retigabine may be related to inhibition of afferent nerve activity and not diminished detrusor contractility.Support or Funding InformationSupported by NIH K01DK103840 to NRT and NIH R37DK053832 to MTN.

Effects of Distigmine on Electrical Field Stimulation-Induced Contraction of Mouse Urinary Bladder Smooth Muscles

Distigmine bromide (distigmine), a reversible carbamate cholinesterase inhibitor, is used in Japan for treating detrusor underactivity, myasthenia gravis, and glaucoma. Although there is clinical evidence about the effectiveness of distigmine in the treatment of detrusor underactivity, mechanisms by which distigmine restores impaired urinary bladder smooth muscle (UBSM) contractility have not been fully investigated. The aims of this study were to investigate the potentiating effects of distigmine on UBSM contractions in response to parasympathetic nerve stimulation induced by electrical field stimulation (EFS) in mice. In isolated mouse UBSM, EFS (1-16 Hz) produced tetrodotoxin-sensitive, frequency-dependent contractions. The contractile responses to EFS were largely attenuated by atropine (10^-6 mol/l). UBSM contractility that occurred in the presence of atropine was nearly eliminated by the addition of α,β-methylene adenosine triphosphate (α,β-mATP, 10^-4 mol/l). Distigmine (3 × 10^-7 mol/l) significantly potentiated EFS-induced contractile responses engendered in the presence of α,β-mATP (10^-4 mol/l), but not atropine (10^-6 mol/l). These findings indicate that distigmine powerfully potentiates UBSM contractions selectively induced by parasympathetic nerve-derived acetylcholine, thereby demonstrating a potential mechanism by which it stimulates detrusor contractile function.

Inhibitory Effects of Antidepressants on Acetylcholine-Induced Contractions in Isolated Guinea Pig Urinary Bladder Smooth Muscle

Background/Aims: To investigate the potential inhibitory effects of 18 clinically available antidepressants on acetylcholine (ACh)-induced contractions in guinea pig urinary bladder smooth muscle (UBSM) in order to predict whether they may induce voiding impairment. Methods: Concentration-response curves for ACh-induced contractions in guinea pig UBSM strips were obtained in the absence or presence of selected antidepressants. When inhibitory effects indicated competitive antagonism, pA₂ values against ACh were calculated and compared to plausible antidepressant blood concentrations. Results: ACh-induced contraction was antagonized competitively within clinical dose ranges by tricyclic antidepressants (imipramine, amitriptyline, trimipramine, clomipramine, nortriptyline, and amoxapine), maprotiline (a tetracyclic antidepressant), and mirtazapine (a noradrenergic and specific serotonergic antidepressant). ACh-induced contraction was also significantly inhibited by mianserin (a tetracyclic antidepressant), paroxetine and sertraline (serotonin-selective reuptake inhibitors, SSRIs), and duloxetine (a serotonin noradrenaline (norepinephrine) reuptake inhibitor, SNRI), albeit at concentrations that substantially exceeded clinically achievable blood levels. However, ACh-induced contractions were not significantly affected by fluvoxamine and escitalopram (SSRIs), milnacipran (an SNRI), trazodone (a serotonin 5-HT_2A receptor antagonist), sulpiride (a dopamine D₂ receptor antagonist), or aripiprazole (a dopamine partial agonist). Conclusion: These findings suggest that in addition to tricyclics, some relatively novel antidepressants such as mirtazapine can induce voiding impairment, attributed to diminished UBSM contractility from the inhibition of muscarinic receptors in the UBSM.

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.

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.

Constitutive PKA activity is essential for maintaining the excitability and contractility in guinea pig urinary bladder smooth muscle: role of the BK channel.

The elevation of protein kinase A (PKA) activity activates the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels in urinary bladder smooth muscle (UBSM) cells and consequently attenuates spontaneous phasic contractions of UBSM. However, the role of constitutive PKA activity in UBSM function has not been studied. Here, we tested the hypothesis that constitutive PKA activity is essential for controlling the excitability and contractility of UBSM. We used patch clamp electrophysiology, line-scanning confocal and ratiometric fluorescence microscopy on freshly isolated guinea pig UBSM cells, and isometric tension recordings on freshly isolated UBSM strips. Pharmacological inhibition of the constitutive PKA activity with H-89 or PKI 14-22 significantly reduced the frequency and amplitude of spontaneous transient BK channel currents (TBKCs) in UBSM cells. Confocal and ratiometric fluorescence microscopy studies revealed that inhibition of constitutive PKA activity with H-89 reduced the frequency and amplitude of the localized Ca(2+) sparks but increased global Ca(2+) levels and the magnitude of Ca(2+) oscillations in UBSM cells. H-89 abolished the spontaneous transient membrane hyperpolarizations and depolarized the membrane potential in UBSM cells. Inhibition of PKA with H-89 or KT-5720 also increased the amplitude and muscle force of UBSM spontaneous phasic contractions. This study reveals the novel concept that constitutive PKA activity is essential for controlling localized Ca(2+) signals generated by intracellular Ca(2+) stores and cytosolic Ca(2+) levels. Furthermore, constitutive PKA activity is critical for mediating the spontaneous TBKCs in UBSM cells, where it plays a key role in regulating spontaneous phasic contractions in UBSM.

Prostaglandin E2 inhibits the large‐conductance voltage‐ and Ca2+‐activated K+ channels in guinea pig urinary bladder smooth muscle (865.7)

Prostaglandin E2 (PGE2) is an important signaling molecule that has been shown to be involved in the regulation of urinary bladder smooth muscle (UBSM) contractility in both rodents and humans. However, the underlying mechanism by which PGE2 evokes UBSM contractions is not well understood. Here, we investigated whether large conductance voltage‐ and Ca2+‐activated K+ (BK) channels mediate the stimulatory effects induced by PGE2 on guinea pig UBSM contractility. We used a combined experimental approach including patch‐clamp electrophysiology, live‐cell Ca2+ imaging, and isometric UBSM tension recordings to investigate BK channel regulation by PGE2 at both cellular and tissue levels. PGE2 increased the spontaneous phasic contractions of UBSM isolated strips in a concentration‐dependent manner (10 nM‐10 µM). BK channel inhibition with paxilline attenuated the PGE2‐induced UBSM phasic contractions, suggesting that BK channels are among the targets of PGE2. Our data also indicate that PGE2 (10 µM) caused an inhibition of the amplitude and frequency of spontaneous transient BK currents and increased the intracellular Ca2+ levels in freshly‐isolated UBSM cells. Thus, for the first time we have revealed a novel mechanism by which BK channels mediate the PGE2 stimulatory effects on guinea pig UBSM contractions.Grant Funding Source: Supported by NIH R01 DK084284 grant to Georgi V. Petkov.

Pharmacological dissection reveals functional roles for KCNQ channel subtypes in human urinary bladder smooth muscle contractility (1057.12)

This study is aimed at investigating the molecular expression of voltage‐gated potassium (KCNQ) channels in urinary bladder smooth muscle (UBSM) and to reveal their functional role in regulating the contractility of this tissue. Using native, freshly‐isolated human UBSM single cells and whole UBSM tissue, we examined the mRNA and protein expression for all KCNQ channel subtypes and elucidated their functional role in human UBSM contractility using RT‐PCR, western blot, and isometric UBSM tension recordings of isolated human UBSM strips. Human UBSM tissues were collected from open bladder surgeries and represent patients without preoperative symptoms of overactive bladder. Expression of KCNQ channel subtypes were identified at both the mRNA and protein levels in human UBSM. Functional studies revealed that spontaneous phasic contractility was significantly increased by the KCNQ channel inhibitor, XE991. On the contrary, the KCNQ channel activators retigabine, L‐364,373 (R‐L3), and ICA‐069673 attenuated UBSM spontaneous phasic contractions. This study demonstrates the novel findings that KCNQ channels are functionally expressed in human UBSM, whereby they serve an important functional role in regulating the spontaneous contractility of this tissue.Grant Funding Source: Supported by NIH R01 DK084284 to Georgi V. Petkov

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.

Functional implications of Kv7 channels expression in guinea pig urinary bladder smooth muscle contractility

The physiological roles of Kv7 channel family have been well studied in various tissues. However, knowledge about the functional role of Kv7 channels in the urinary bladder smooth muscle (UBSM) is limited. We investigated Kv7 channels molecular expression in guinea pig UBSM and further assessed their functional role using isometric DSM tension recordings and Kv7 channels modulators. RT‐PCR experiments revealed mRNA expression for Kv7.1, Kv7.2, Kv7.3, Kv7.4, and Kv7.5 channel subunits in guinea pig UBSM tissue. Quantitative PCR analysis showed that in UBSM tissue, Kv7 channel subunits relative mRNA expression level was as follows: Kv7.1 ≈ Kv7.2 >; Kv7.3 ≈ Kv7.5 >; Kv7.4. Retigabine, a Kv7 channel activator, and L‐364,373, a selective Kv7.1 channel activator, induced relaxation of spontaneous phasic contraction amplitude, force integral, duration, and frequency as well as the 10 Hz electrical field stimulation (EFS)‐induced contractions amplitude and force of UBSM isolated strips (IC50 = 3 – 6 μM). Linopiridine and XE991, two well‐established Kv7 channel inhibitors, increased the spontaneous phasic contraction amplitude and force as well as the 10 Hz EFS‐induced contractions amplitude and force of UBSM isolated strips (IC50 = 3 – 4 μM). In summary, we revealed the expression and the regulatory role of Kv7 channels in the spontaneous and nerve‐evoked contractions of guinea pig UBSM. Supported by NIH DK084284 to GVP.

Changes in Urinary Bladder Smooth Muscle Contractility and the Role of Rho Kinase and Isoprostane after Partial Bladder outlet Obstruction in rats

Objective: Bladder outlet obstruction is a common urological problem that associatedwith many cellular changes and remodeling of urinary bladder detrusor smoothmuscle. This study was designed to determine the changes in Rho kinase (ROCK) andisoprostane levels in urinary bladder homogenate after partial bladder outletobstruction (PBOO); and to evaluate the contractile response of isolated rat detrusormuscle after PBOO. Methods: The present work was carried out on 30 male albinorats that were divided into 3 groups (10 rats/group): group I: consisted of normalrats, they were sham operated and considered as control group, group II: consisted ofrats that were undergone PBOO for 14days, group III: consisted of rats that wereundergone PBOO for 28 days. The following parameters were assessed in rats of allgroups: urinary bladder weight, levels of both Rho kinase and isoprostane in thetissues of urinary bladder as well as the contractile response of detrusor muscle bothspontaneously and in response to acetyl choline. Results: The present study revealeda significant increase in the weight of urinary bladder in rats of PBOO of group IIand III as compared to sham operated control of group I. There was a significantincrease in the mean value of Rho kinase and isoprostane in the tissue of the urinarybladder after obstruction. 14 days after obstruction was associated with a significantincrease in the mean value of Rho kinase which was significantly high compared togroup I (sham operated rats) and group III (28days of obstruction), 28 days ofobstruction was associated with decrease in the mean value of Rho kinase comparedto 14 days of obstruction, however, its value was above the control. Regarding thecontractile response, PBOO was associated with increased spontaneous activity ofdetrusor muscle and increased tonic response to acetyl choline which wassignificantly high after 14 and 28 days of obstruction compared to the control.Conclusion: The results of the present study demonstrated that Rho kinase andisoprostane levels were elevated after bladder outlet obstruction and that Rho kinasecould play a crucial role in the regulation of rat urinary bladder smooth musclecontraction and tone. Therefore, Rho kinase inhibitors may prove to betherapeutically useful in the treatment of lower urinary tract disorders associatedwith changes in the physiology of bladder smooth muscle contractility.

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+).

Inhibition of phosphodiesterases relaxes urinary bladder smooth muscle via activation of the large conductance voltageand Ca2+‐activated K+ channels

Our previous studies showed that activation of cAMP signaling pathways reduces urinary bladder smooth muscle (UBSM) contractility by increasing the large conductance Ca2+‐activated K+ (BK) channel activity. Utilizing isometric UBSM tension recordings and the perforated patch‐clamp technique, we tested the hypothesis whether inhibition of phosphodiesterases (PDEs) can reduce guinea pig UBSM excitability and contractility by increasing BK channel activity. Inhibition of PDEs by 3‐isobutyl‐1‐methylxanthine (IBMX) concentration‐dependently reduced UBSM spontaneous and carbachol‐induced phasic contractions. IBMX also reduced electrical field stimulation (0.5–50 Hz)‐induced contractions of UBSM strips. Blocking BK channels with paxilline diminished the inhibitory effects of IBMX on UBSM contractility. IBMX increased the transient BK currents (TBKCs) frequency by ~3‐fold. However, IBMX did not affect the depolarization‐induced steady‐state whole‐cell BK currents. IBMX increased the frequency of the spontaneous transient hyperpolarizations by ~2‐fold and hyperpolarized the UBSM cell resting membrane potential by ~6 mV. Blocking the BK channels abolished the IBMX effect on the resting membrane potential. Our data reveal that PDEs inhibition with IBMX relaxes guinea pig UBSM via TBKCs activation and subsequent UBSM cell membrane hyperpolarization.Supported by NIH DK084284 to G.V.P.

Voltage-gated K+ channels sensitive to stromatoxin-1 regulate myogenic and neurogenic contractions of rat urinary bladder smooth muscle

Members of the voltage-gated K(+) (K(V)) channel family are suggested to control the resting membrane potential and the repolarization phase of the action potential in urinary bladder smooth muscle (UBSM). Recent studies report that stromatoxin-1, a peptide isolated from tarantulas, selectively inhibits K(V)2.1, K(V)2.2, K(V)4.2, and K(V)2.1/9.3 channels. The objective of this study was to investigate whether K(V) channels sensitive to stromatoxin-1 participate in the regulation of rat UBSM contractility and to identify their molecular fingerprints. Stromatoxin-1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and tone in isolated UBSM strips. However, stromatoxin-1 (100 nM) had no effect on the UBSM contractions induced by depolarizing agents such as KCl (20 mM) or carbachol (1 microM). This indicates that, under conditions of sustained membrane depolarization, the K(V) channels sensitive to stromatoxin-1 have no further contribution to the membrane excitability and contractility. Stromatoxin-1 (100 nM) increased the amplitude of the electrical field stimulation-induced contractions, suggesting also a role for these channels in neurogenic contractions. RT-PCR experiments on freshly isolated UBSM cells showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3, but not K(V)4.2 channel subunits. Protein expression of K(V)2.1 and K(V)2.2 channels was detected using Western blot and was further confirmed by immunocytochemical detection in freshly isolated UBSM cells. These novel findings indicate that K(V)2.1 and K(V)2.2, but not K(V)4.2, channel subunits are expressed in rat UBSM and play a key role in opposing both myogenic and neurogenic UBSM contractions.