Human Urinary Bladder Smooth Muscle Research Articles

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.

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

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

Transient Receptor Potential Melastatin 4 Channel Regulation by Inositol Trisphosphate Receptor in Human Urinary Bladder Smooth Muscle

Overactive bladder (OAB) is a highly prevalent and debilitating condition that lacks an effective therapeutic treatment. The contraction and relaxation of urinary bladder smooth muscle (UBSM) facilitates the voiding and storage of urine, the two main functions of the urinary bladder. Some forms of OAB have been directly linked to UBSM dysfunction. Thus, efforts to uncover unknown regulatory mechanisms in UBSM function are urgently needed. Among the potential novel targets for OAB, emerging evidence points to key roles for transient receptor potential melastatin 4 (TRPM4) channels in UBSM excitability and contractility. However, the cellular mechanisms regulating TRPM4 channel activity in human UBSM has been virtually unexplored. As a potential cellular mechanism regulating TRPM4 channel activity in UBSM, we have directed our attention to the inositol trisphosphate (IP3) receptors (IP3Rs), which facilitate the release of Ca2+ from the sarcoplasmic reticulum (SR). Since the TRPM4 channels are activated by Ca2+, IP3R‐mediated Ca2+ release of the SR represents a potential Ca2+ source for TRPM4 channel activation. In accordance with the approved IRB Pro00045232 of the Medical University of South Carolina, we used clinically‐characterized human UBSM tissues from 6 donor patients to investigate the molecular and functional interactions of the TRPM4 channels and IP3Rs in human UBSM. Using in situ proximity ligation assay (PLA) and amphotericin‐B perforated patch‐clamp electrophysiology, we sought to test the hypothesis that TRPM4 channels are tightly associated with the IP3Rs and are activated by IP3R‐mediated Ca2+ release of the SR in human UBSM. As determined by in situ PLA, we demonstrated colocalization of the TRPM4 channels and IP3Rs in human UBSM cells. As the TRPM4 channels and IP3Rs must be located within close apposition to functionally interact, these findings support the concept of a TRPM4‐IP3R regulatory mechanism in human UBSM function. To investigate IP3R regulation of TRPM4 channel activity, we sought to determine the consequences of IP3R pharmacological inhibition on TRPM4 channel‐mediated transient inward cation currents (TICCs). In freshly‐isolated human UBSM cells, the selective IP3R inhibitor xestospongin C significantly decreased TICCs. These findings suggest that the SR IP3Rs have a key role in mediating the Ca2+‐dependent activation of TRPM4 channels in human UBSM. In conclusion, this study provides novel insight into the cellular mechanisms regulating TRPM4 channel activity in human UBSM function. Our study reveals that the TRPM4 channels and IP3Rs are spatially and functionally coupled in human UBSM, information that is critical for further evaluating the potential role of the TRPM4 channels as novel therapeutic targets for OAB.Support or Funding InformationSupported by NIH grant R01‐DK106964 to Georgi V. Petkov and F31‐DK104528 to Aaron Provence.

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.

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.

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

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 & 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.

SK channel-selective opening by SKA-31 induces hyperpolarization and decreases contractility in human urinary bladder smooth muscle

Overactive bladder (OAB) is often associated with increased involuntary detrusor smooth muscle (DSM) contractions during the bladder-filling phase. To develop novel therapies for OAB, it is critical to better understand the mechanisms that control DSM excitability and contractility. Recent studies showed that small-conductance Ca(2+)-activated K(+) (SK) channels, SK3 channels, in particular, regulate human DSM function. However, the concept that SK channel-selective pharmacological activation can decrease the excitability and contractility directly in human DSM needs further exploration. Here, we studied the effect of the novel and potent SK channel activator, SKA-31 (or naphtho [1,2-d]thiazol-2-ylamine), on human DSM excitability and contractility at the cellular and tissue level. We used isometric tension recordings on human DSM-isolated strips and the perforated patch-clamp technique on freshly isolated native human DSM cells. SKA-31 significantly decreased spontaneous phasic contractions of DSM-isolated strips. In the presence of the SK channel blocker, apamin, the inhibitory effects of SKA-31 on the DSM spontaneous phasic contractions were significantly reduced. SKA-31 decreased the carbachol- and KCl-induced contractions in human DSM strips. Electrical field stimulation-induced contractions were significantly attenuated in the presence of SKA-31 at all stimulation frequencies (0.5-50 Hz). SKA-31 hyperpolarized the resting membrane potential of human DSM cells. Apamin abolished the hyperpolarizing effect of SKA-31, indicating the involvement of SK channel activation. These results support the concept that pharmacological activation of SK channels with selective openers may represent an attractive new pharmacological approach for decreasing DSM excitability and contractility, thus controlling OAB.

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+).

Expression and function of KV2-containing channels in human urinary bladder smooth muscle

The functional role of the voltage-gated K(+) (K(V)) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of K(V)2.1, K(V)2.2, and the electrically silent K(V)9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of K(V)2.1, K(V)2.2, and K(V)4.2 homotetrameric channels and of K(V)2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca(2+) imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments 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 in human isolated DSM cells. K(V)2.1 and K(V)2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced K(V) current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca(2+) level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5-30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive K(V)2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

Generation of a human urinary bladder smooth muscle cell line

We report a cell line (hBSM) established from human urinary bladder wall smooth muscle that maintains most of the phenotypic characteristics of smooth muscle cells. Cells were dissociated from the muscular layer with collagenase (1mg/ml) and collected and grown in M199 supplemented with 10% fetal calf serum and 1% antibiotic-antimycotic. Primary cultures were grown for 2d and small colonies were isolated by placing glass rings around the colonies. These colonies were picked up with a fine-tipped Pasteur pipette and subcultured. This procedure was repeated several times until a culture with a uniform stable morphology was obtained. hBSM cells are elongated with tapered ends, and in high density cultures, they form swirls of cells arranged in parallel. These cells have a doubling time of approximately 72 h. Western blotting and immunofluorescence microscopy revealed stable expression of smooth muscle-specific proteins, including myosin isoforms (N-terminal isoforms SM-A/B and C-terminal isoforms SM1/2), SM22, α-smooth muscle actin, h-caldesmon, Ca(2+)-dependent myosin light chain kinase, and protein kinase G. These cells contract upon exposure to 10μM bethanechol and this contraction is reversible by washing away the drug. Karyotyping showed tetraploidy with a modal chromosome number of 87, with multiple rearrangements. To our knowledge, the hBSM cell line is the first human cell line established from bladder wall smooth muscle that expresses both N- and C-terminal smooth muscle myosin isoforms. This cell line will provide a valuable tool for studying transcriptional regulation of smooth muscle myosin isoforms and effects of drugs on cellular function.

BK channel activation with NS1619 decreases excitability and contractility of human urinary bladder smooth muscle

Overactive bladder syndrome is frequently associated with increased detrusor smooth muscle (DSM) contractility. We explored if activation of the large conductance Ca2+-activated K+ (BK) channel with NS1619, a selective BK channel opener, can reduce the excitability and contractility of human DSM. We used the amphotericin perforated whole cell patch-clamp technique and isometric tension recordings of freshly isolated human DSM obtained from open bladder surgeries. In current-clamp mode, NS1619 (30 μM) hyperpolarized the resting membrane potential by ~3 mV. This effect was reversed by iberiotoxin (200 nM), a selective BK channel inhibitor. NS1619 (30 μM) significantly increased the amplitude of the whole-cell BK currents at all stimulating voltages from 0 to +80 mV. BK channel activation with NS1619 (30 μM) significantly inhibited the amplitude, muscle force, frequency, duration, and tone of the spontaneous and pharmacologically-induced contractions of isolated human DSM strips. The amplitude of neurogenic contractions evoked by electrical field stimulation (0.5–50 Hz) was significantly inhibited by NS1619 (30 μM). Iberiotoxin (200 nM) suppressed the inhibitory effects of NS1619. Our data suggest that pharmacological activation of BK channel could represent a novel treatment option for overactive bladder and urinary incontinence. Supported by NIH DK084284 and DK083687 to Georgi V. Petkov.

Effect of the SK/IK channel modulator 4,5‐dichloro‐1,3‐diethyl‐1,3‐dihydro‐benzoimidazol‐2‐one (NS4591) on contractile force in rat, pig and human detrusor smooth muscle

• To investigate the importance of small (SK)- and intermediate (IK)-conductance Ca2(+) -activated K(+) channels on bladder function, by studying the effects of 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591), a new modulator of SK/IK channels, on contractions induced by electrical field stimulation (EFS) and carbachol in rat, pig and human detrusor. • Detrusor biopsies were obtained from rats, pigs and male patients undergoing cystectomy because of bladder cancer. • Force was recorded using myographs. • Intracellular free Ca(2+) was measured in myocytes using microfluorimetry. • In rat bladder rings subjected to EFS, cumulative addition of NS4591 (0.1-30 µM) decreased force by 82 ± 2.9% (n = 6).This effect was reduced by 64 ± 5.2% in the presence of 0.3 µM apamin, a specific inhibitor of SK channels. Apamin increased the force evoked by EFS significantly: force was increased by 14.2 ± 3.4% (n = 5) and 10.1 ± 2.6% (n = 7) in pig and human detrusor strips, respectively (P = 0.04 and P = 0.02). • The cumulative addition of NS4591 (0.3-30 µM) significantly reduced the amplitude of carbachol-induced rhythmic oscillations by 62.0 ± 12.0% (n = 12) and the minimum force between oscillations by 30 ± 5% (n = 9) in pig detrusor strips (P < 0.005). In the presence of 10 µM NS4591, carbachol (1 µM) induced rhythmic contractions with an amplitude and normalized mean power frequency (nmeanPF) of 8.4 ± 5.1% and 0.11 ± 0.06 mN root mean square (rms) Hz (n = 12), respectively, vs. 21 ± 3.4% and 0.17 ± 0.04 mN rms Hz in control strips (n = 13). Apamin induced 6- and 11-fold increases in amplitude and nmeanPF vs. 1.3- and 2-fold increases in control strips. • In human detrusor strips (n = 15), the cumulative addition of NS4591 (1-30 µM) significantly reduced the amplitude by 69 ± 11%, the nmeanPF by 78 ± 6% and the minimum force between carbachol-induced oscillations by 59 ± 5% (P < 0.008). The addition of apamin (0.3 µM) before application of 1 µM carbachol abolished the effects of NS4591 on amplitude and partially abolished its effect on nmeanPF by 41 ± 7%, vs. a 78 ± 6% reduction in the absence of apamin (n = 8). • In spontaneously active detrusor preparations, NS4591 reduced or abolished contractions. • Furthermore, NS4591 (10 µM) decreased the carbachol-induced increase in the fura-2 ratio by 43 ± 3% compared with control (n = 12) (P < 0.03). • The SK/IK channel modulator NS4591 inhibits EFS- and carbachol-induced contractions in rat, pig and human detrusor muscle. • NS4591 may have therapeutic potential for treatment of detrusor overactivity.

Human urinary bladder smooth muscle is dependent on membrane cholesterol for cholinergic activation

Voiding is mediated by muscarinic receptors in urinary bladder smooth muscle cells. Lipid rafts and caveolae are cholesterol enriched membrane domains that modulate the activity of G protein-coupled receptors and second messenger systems. Conflicting findings regarding sensitivity of muscarinic signalling to cholesterol desorption, which perturbs lipid rafts and caveolae, have been reported, and no study has used human urinary bladder. Here, the dependence of human bladder muscarinic receptor signalling on plasma membrane cholesterol was examined. Nerve-mediated contraction, elicited by electrical field stimulation of human bladder strips, was impaired by desorption of cholesterol using methyl-beta-cyclodextrin, and the concentration-response curve for the muscarinic agonist carbachol was right-shifted. No effect of cholesterol desorption was observed in rat, and in mouse increased maximum contraction was seen. Expression of caveolin-1, PLCbeta1 and M3 muscarinic receptors did not differ between species in a manner that would explain the differential sensitivity to cholesterol desorption. In human bladder, threshold depolarisation eliminated the difference between cyclodextrin-treated and control preparations. Contraction elicited by depolarisation per se was not affected. M3 muscarinic receptors appeared clustered along plasma membrane profiles as shown by immunohistochemical staining of human bladder, but no redistribution in association with cholesterol reduction was seen. Thus, muscarinic receptor-induced contraction of the urinary bladder exhibits species-specific differences in its sensitivity to cholesterol desorption suggesting differential roles of lipid rafts/caveolae in muscarinic receptor signalling between species.

Involvement of β 3-adrenoceptors in mouse urinary bladder function: Role in detrusor muscle relaxation and micturition reflex

β 3-adrenoceptor activation produces relaxation of human urinary bladder smooth muscle (detrusor). Therefore, β 3-adrenoceptor agonism is being investigated as a new therapeutic strategy for the treatment of overactive bladder. The aim of the current study was to identify the functional presence of β 3-adrenoceptors in mouse isolated urinary bladder using the selective β 3-adrenoceptor agonist CL316,243 and antagonists SR59230A and L748,337. The effects of CL316,243 on basal tone, spontaneous activity and electrical field stimulation (EFS)-induced contractions were investigated using in vitro techniques, while the in vivo effects of intravenously administered CL316,243 on the micturition reflex were investigated using cystometry. CL316,243 decreased basal tone (pEC 50 = 6.4 ± 0.4) as well as spontaneous activity (53 ± 7% at 3 µM) and inhibited EFS-induced contractions (pEC 50 = 7.0 ± 0.2) of the detrusor muscle. The β 3-adrenoceptor antagonist SR59230A (1 µM) significantly inhibited the relaxing effects of CL316,243 on basal tone and neurogenic contractions (pA 2 = 7.0 and 7.2, respectively). Another β 3-adrenoceptor antagonist L748,337 (1-10 µM) significantly blocked the CL316,243-evoked inhibition of neurogenic contractions in a concentration-dependent manner (pK B = 6.8), while the selective β 2-adrenoceptor antagonist ICI118,551(30 nM) had no effect. In anesthetized mice, CL316,243 (0.03 and 0.1 mg/kg, i.v.) significantly increased bladder capacity and threshold pressure without a modification of bladder compliance. Moreover, it induced a significant decrease in the amplitude of both micturition and non-voiding contractions. Based on the current results obtained using the β 3-adrenoceptor agonist CL316,243 (as well as various β-adrenoceptor antagonists), functional β 3-adrenoceptors appear to be present in mouse urinary bladder.

The large conductance Ca2+‐ and voltage‐activated K+ channel is the primary K+ channel that controls cell excitability and contractility in human urinary bladder smooth muscle

We examined the large conductance Ca2+‐activated K+ (BK) channel expression, electrophysiological properties, and possible physiological roles in human urinary bladder smooth muscle (UBSM). RT‐PCR with freshly isolated human UBSM cells shows mRNA expression of BK channel pore‐forming α, and regulatory β1/β4 subunits. The expression of all three subunits was confirmed by specific antibodies and immunocytochemistry on single cells. The majority of the whole‐cell outward current induced by voltage‐step depolarizations (‐40 to +80 mV) was inhibited by iberiotoxin (IBTX), a BK channel selective inhibitor. Voltage‐dependent spontaneous transient outward currents (STOCs) were recorded from ‐40 to +40 mV. STOCs were blocked by IBTX or ryanodine. In current‐clamp mode, IBTX caused ~3 mV depolarization. In whole cell configuration, in the absence of STOCs, single channels of 139.5 pS conductance with voltage‐dependent amplitude and open probability were recorded. These channels were blocked by IBTX, consistent with the BK channel. In isolated strips, IBTX increased spontaneous phasic contraction amplitude, force, and tone as well as the amplitude of electrical field induced contractions. In conclusion, the BK channel in the human bladder consists of α, β1 and β4 subunits and is the primary K+ channel that controls cell excitability and contractility of human UBSM.Supported by NIH DK‐070909 to G.V. Petkov.

Functional and electrophysiological identification of large conductance voltage‐ and Ca2+‐activated K+ (BK) channels in human urinary bladder smooth muscle

The large conductance voltage‐ and Ca2+‐activated K+ (BK or KCa1.1) channel is a key player in controlling excitability and contractility of animal urinary bladder smooth muscle (UBSM), however its role in human UBSM is unknown. Here, using the BK channel specific inhibitor‐iberiotoxin, functional studies and the perforated patch‐clamp technique, we identified the BK channel in human UBSM. In isolated UBSM strips iberiotoxin increased spontaneous phasic contraction amplitude and force (n=10). In isolated UBSM cells, voltage‐step depolarizations caused large K+ currents (∼1 nA; n=15). About 80% of these K+ currents were inhibited by iberiotoxin. In the voltage range of −40 to +40 mV, these cells also revealed spontaneous transient outward currents (STOCs). STOCs amplitude and frequency increased with increasing the holding potential toward positive values (n=20). STOCs were inhibited by iberiotoxin (n=4). In the presence of ryanodine, when the STOCs were blocked, single channels with large single channel conductance (120.6 pS) were observed (n=10). Their amplitude and open probability were voltage‐dependent. In current‐clamp mode, iberiotoxin caused membrane potential depolarization (n=4). The data reveal that the BK channel is the major voltage‐dependent K+ channel expressed in human UBSM cells that plays a key role in determining cell excitability and contractility. Supported by DK070909 to G.V.P.

Potentiation of Potassium Currents by Beta-Adrenoceptor Agonists in Human Urinary Bladder Smooth Muscle Cells: A Possible Electrical Mechanism of Relaxation

We examined the effects of β-adrenoceptor agonists on the membrane currents of smooth muscle cells from the human urinary bladder using a whole-cell patch clamp to investigate the involvement of Ca²⁺-activated K⁺ (K_Ca) channels in relaxation by β-adrenergic agonists. With 0.05 mmol/l EGTA in the patch pipette, depolarizing pulses evoked outward rectifying currents. Isoproterenol (1 µmol/l) significantly increased the membrane currents by 75% at +80 mV with 0.05 mmol/l EGTA pipette solution. BRL 37344 (1 µmol/l) significantly increased the membrane currents by 44% at +80 mV. Iberiotoxin (100 nmol/l) significantly decreased the membrane currents by 60% at +80 mV. In the presence of iberiotoxin, the potentiation of the outward currents by isoproterenol was greatly suppressed and, in the presence of iberiotoxin and apamin (1 µmol/l), the potentiation by isoproterenol was totally abolished. On the other hand, with 5 mmol/l EGTA pipette solution, depolarizing pulses evoked smaller outward currents. Isoproterenol (1 µmol/l) did not change the membrane currents with 5 mmol/l EGTA pipette solution. The real-time PCR analysis revealed the expression of β₂-adrenoceptors in the cells. These results suggest that Ca²⁺-activated and iberiotoxin- and apamin-sensitive currents via both large-conductance and small-conductance K_Ca channels could be increased by stimulation of β₂-adrenoceptors.