Urinary Bladder Smooth Muscle Function Research Articles

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.

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.

Testosterone regulates urinary bladder smooth muscle function through a signaling mechanism involving direct activation of large conductance voltage‐ and Ca2+‐activated K+ channels

In addition to improving sexual function, testosterone replacement therapy has been reported to have beneficial effects in ameliorating lower urinary tract symptoms (LUTS) by increasing bladder capacity, compliance, and decreasing bladder pressure. The cellular mechanism by which testosterone regulates urinary bladder smooth muscle (UBSM) excitability have not been elucidated. Here, we provided a detailed investigation into the regulatory role of testosterone on UBSM excitability in freshly‐isolated guinea pig UBSM cells. We used amphotericin‐B perforated patch‐clamp, which preserves the native intracellular environment of UBSM cells intact, as well as single channel recordings in inside‐out excised membrane patches. Our patch‐clamp data showed that testosterone (100 nM) significantly increased the depolarization‐induced whole cell outward currents in freshly‐isolated guinea pig UBSM cells (n=14). The effect of testosterone was more pronounced at positive membrane potentials, and at +80 mV testosterone increased the current density by ~19% in comparison to control (n=14). The majority of the whole cell outward currents in UBSM cells are conducted by the large conductance voltage‐ and Ca2+‐activated K+ (BK) channels. These channels are key regulators of UBSM excitability and contractility, and modulating their activity could be a basis for the development of promising novel therapies for bladder dysfunction. Our data revealed that selective pharmacological inhibition of the BK channels with paxilline (1 μM) completely abolished the stimulatory effect of testosterone on the whole cell outward currents (n=6), suggesting that testosterone activates the whole‐cell currents due to BK channel stimulation rather than other mechanisms. In UBSM, localized Ca2+ release from sarcoplasmic reticulum activate BK channels and generate transient BK currents (TBKCs). Our data revealed that at holding potential of −20 mV, UBSM cells exhibit TBKC activity which was significantly increased by testosterone (100 nM) with ~27% in comparison to control (n=5). These results provided additional line of evidence for the stimulatory role of testosterone on BK channels in UBSM cells. Furthermore, our current‐clamp recordings revealed that testosterone (100 nM) hyperpolarized the UBSM cell resting membrane potential by ~2.5 mV, consistent with the reported hyperpolarizing effects of BK channel activators in this cell type (n=24). To investigate the intracellular signaling mechanism by which testosterone regulates BK channels, we performed a series of single BK channel recordings in excised membrane patches from UBSM cells. The data showed that testosterone (100 nM) increased the single BK channel open probability by more than 300% in comparison to control (n=9). These results support the concept that in UBSM cells testosterone can activate BK channels through a direct non‐genomic mechanism. In conclusion, we provide compelling mechanistic evidence that under physiological conditions, testosterone at nanomolar concentrations directly activates BK channels in guinea pig UBSM cells, and thus regulate UBSM excitability.Support or Funding InformationSupported by NIH grant R01 DK106964 to Georgi V. Petkov.

Sexually dimorphic effects of early life stress in rat pups on urinary bladder detrusor muscle contractility in adulthood.

BackgroundPainful bladder syndrome/interstitial cystitis (PBS/IC) is a chronic disorder that is commonly seen in women who report a history of adversity in early life. Here, we test the hypothesis that early life stress (ELS) induces sexually dimorphic abnormalities in urinary bladder smooth muscle function in adulthood.MethodsMale and female rat pups were conditioned on postnatal (PN) days 8–12 with either a “predictable or “unpredictable” odor-shock, or odor only control treatment. In adulthood, urinary bladder function was assessed in vivo via urine spot analysis and in vitro via contractile responses to electrical field stimulation (EFS) and membrane depolarization with potassium chloride (KCl).ResultsIn adulthood, we found that female rats exposed to unpredictable ELS showed a significant (p < 0.05) increase in urine voiding volume compared to predictable ELS or controls. We also found that detrusor muscle contractile responses to EFS were significantly (p < 0.001) decreased following unpredictable ELS in adult female rats compared to the predictable ELS or controls. In male rats exposed to ELS, there was no difference in voiding volume or EFS-induced contractility between groups. In adulthood, the myogenic smooth muscle response to KCl was not significantly different between groups. Histological analysis from adult female and male rats revealed no differences in the appearance of the urinary bladder in rats exposed to ELS.ConclusionsIn summary, our findings provide evidence to support abnormalities in the nerve-mediated contractile responses of the detrusor smooth muscle in adult female rats following ELS. We speculate that these sexually dimorphic alterations in urinary bladder function may account, at least in part, for the female predominance of PBS/IC.

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.

TRPM4 channel: a new player in urinary bladder smooth muscle function in rats

The TRPM4 channel is a Ca(2+)-activated, monovalent cation-selective channel of the melastatin transient receptor potential (TRPM) family. The TRPM4 channel is implicated in the regulation of many cellular processes including the immune response, insulin secretion, and pressure-induced vasoconstriction of cerebral arteries. However, the expression and function of the TRPM4 channels in detrusor smooth muscle (DSM) have not yet been explored. Here, we provide the first molecular, electrophysiological, and functional evidence for the presence of TRPM4 channels in rat DSM. We detected the expression of TRPM4 channels at mRNA and protein levels in freshly isolated DSM single cells and DSM tissue using RT-PCR, Western blotting, immunohistochemistry, and immunocytochemistry. 9-Hydroxyphenanthrene (9-phenanthrol), a novel selective inhibitor of TRPM4 channels, was used to examine their role in DSM function. In perforated patch-clamp recordings using freshly isolated rat DSM cells, 9-phenanthrol (30 μM) decreased the spontaneous inward current activity at -70 mV. Real-time DSM live-cell Ca(2+) imaging showed that selective inhibition of TRPM4 channels with 9-phenanthrol (30 μM) significantly reduced the intracellular Ca(2+) levels. Isometric DSM tension recordings revealed that 9-phenanthrol (0.1-30 μM) significantly inhibited the amplitude, muscle force integral, and frequency of the spontaneous phasic and pharmacologically induced contractions of rat DSM isolated strips. 9-Phenanthrol also decreased the amplitude and muscle force integral of electrical field stimulation-induced contractions. In conclusion, this is the first study to examine the expression and provide evidence for TRPM4 channels as critical regulators of rat DSM excitability and 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+).

Changes in urinary bladder smooth muscle function in response to colonic inflammation

Changes in urinary bladder smooth muscle function in response to colonic inflammation

Changes in urinary bladder smooth muscle function in response to colonic inflammation

Visceral organ "cross talk" is suspected to contribute to multiorgan symptomatology found in conditions such as irritable bowel syndrome and interstitial cystitis. The goal of the present study was to investigate the short- and long-term effects of acute colitis on bladder detrusor muscle contractility. We hypothesized that inflammation of the colon leads to changes in bladder function via direct changes in detrusor smooth muscle contractility. In this study, colonic inflammation was induced in male rats via an enema of trinitrobenzenesulfonic acid (TNBS) (50 mg/kg, 0.5 ml, 25% ethanol). Colitis was confirmed using gross morphology, histology, and measurements of myeloperoxidase activity. Saline enema-treated rats served as controls. Three, 15, and 30 days postenema treatment, bladder detrusor muscle contractility was investigated in response to electrical field stimulation (EFS), cholinergic agonism with carbachol (CCh), and KCl. During active colonic inflammation (day 3 post-TNBS enema), the bladder detrusor muscle appeared normal and showed no significant inflammation. However, abnormalities in bladder detrusor muscle contractility occurred in response to EFS and CCh but not KCl. During and after recovery from colonic inflammation (days 15 and 30 post-TNBS enema), changes in bladder detrusor muscle contractility in response to EFS and CCh returned to control levels. We found that a transient colonic inflammatory insult significantly attenuates the amplitude of bladder detrusor muscle contractions in vitro, at least in part, through changes in cholinergic innervation, which are reversible after recovery from the colitis.

Overactive Bladder and Incontinence in the Absence of the BK Large Conductance Ca2+-activated K+ Channel

BK large conductance voltage- and calcium-activated potassium channels respond to elevations in intracellular calcium and membrane potential depolarization, braking excitability of smooth muscle. BK channels are thought to have a particularly prominent role in urinary bladder smooth muscle function and therefore are candidate targets for overactive bladder therapy. To address the role of the BK channel in urinary bladder function, the gene mSlo1 for the pore-forming subunit of the BK channel was deleted. Slo(-/-) mice were viable but exhibited moderate ataxia. Urinary bladder smooth muscle cells of Slo(-/-) mice lacked calcium- and voltage-activated BK currents, whereas local calcium transients ("calcium sparks") and voltage-dependent potassium currents were unaffected. In the absence of BK channels, urinary bladder spontaneous and nerve-evoked contractions were greatly enhanced. Consistent with increased urinary bladder contractility caused by the absence of BK currents, Slo(-/-) mice demonstrate a marked elevation in urination frequency. These results reveal a central role for BK channels in urinary bladder function and indicate that BK channel dysfunction leads to overactive bladder and urinary incontinence.

Effect of partial outlet obstruction on rabbit urinary bladder smooth muscle function.

Bladder outlet obstruction secondary to benign prostate hyperplasia is associated with many cellular changes. This study was designed to determine whether these changes involve the contractile apparatus. Bladder smooth muscles from rabbits subjected to partial outlet obstruction for 2 wk were mounted for isometric force, isotonic shortening velocity, and myosin light chain (MLC) phosphorylation levels. Muscle strips from obstructed bladders exhibited spontaneous phasic activity; muscle strips from control bladders did not. Muscle strips from obstructed bladders exhibited increased sensitivity and higher levels of stress in response to the cumulative addition of KCl or carbachol compared with control. During noncumulative addition of KCl or carbachol, no differences in sensitivity were noted. Muscle strips from obstructed bladders had elevated basal MLC phosphorylation levels and stimulation produced small increases in MLC phosphorylation compared with control. V(max) during KCl stimulation of muscle strips from obstructed bladders was 10-fold lower than control. Our results suggest that bladder outlet obstruction produces a muscle cell that develops higher levels of force but with greatly reduced cross bridge cycling rates.

Effects of bethanechol on canine urinary bladder smooth muscle function

We studied whether the effects of bethanechol are mediated via a muscarinic receptor, the role of extracellular calcium on bladder contraction, and down-regulation of bladder contraction by bethanechol after activation with potassium chloride (KCl) and acetylcholine (Ach). Smooth muscle strips of normal urinary bladder were studied with standard methods to measure isometric force. Bethanechol caused a dose-dependent increase in bladder contraction. The potency of bethanechol is higher than Ach, as shown by higher peak active isometric stress (Pmax) and lower half-maximal contraction (ED50) (P<0.01). The contractile responses to bethanechol were diminished in the presence of atropine, nifedipine and in calcium-free medium as shown by Pmaxdecreased by 58%, 87% and 65% andED50 increased by 314-, 24- and 16-fold, respectively. When bladder strips were stimulated with KCl and Ach, pre-treatment with bethanechol reduced the responses to KCl by 116–242% (P<0.05), while the contractile responses to Ach were unaltered. Thus, bethanechol induces bladder contraction via muscarinic receptor activation while both intracellular and extracellular calcium play a crucial role on bladder smooth muscle contraction. The mechanisms of down-regulation by bethanechol may be related to interference with calcium influx into the smooth muscle cells, rather than the desensitisation of muscarinic receptors or post-receptor steps of signal transduction following bethanechol binding to the receptor.