Urinary Bladder Smooth Muscle Strips 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.

The mast cell activator Compound 48/80 causes phasic urinary bladder smooth muscle contractions independent of histamine release

Inflammatory mediators released from mast cells cause both transient and phasic contractions of urinary bladder smooth muscle (UBSM). These contractions are viewed as initial disruptors of normal bladder function and can lead to profound bladder pathologies. However, the mechanisms responsible for each of these contractions has not been thoroughly investigated. Previously, we discovered that histamine-mediated contractions rapidly desensitize, whereas the mast cell activator compound 48/80 (10 µg/ml) caused both a long-lasting increase in the amplitude and frequency of phasic contractions in UBSM strips as well as a transient increase in baseline tension. Thus, we hypothesized that phasic contractions caused by compound 48/80 depended on the release of histamine from mast cells. Isometric contractility was performed with urothelium-intact UBSM strips from C57BL/6 mice in the presence or absence of the following drugs: the cholinergic antagonist atropine (2 µM); the 5HT2A receptor antagonist MDL 11939 (1 µM); the histamine H1 receptor antagonist fexofenadine (10 µM), the histamine H2 receptor antagonist roxatidine (25 µM), the H2 antagonist cimetidine (10 µM); or the mast cell stabilizer cromolyn (100 µM). None of the compounds tested significantly reduced the amplitude and duration of the spontaneous phasic contractions caused by compound 48/80. However, both MDL 11939 and cromolyn significantly reduced the baseline contraction. Our data suggest that responses to the mast cell activator compound 48/80 are a superimposition of 2 components: (1) a slowly desensitizing increase in baseline tension caused by release of 5-HT from mast cells, and (2) large-amplitude phasic contractions that are histamine- and 5HT-independent. However, the mechanism responsible for increases in the amplitude of phasic contractions remains unclear.

Identification and Characterization of a Novel Large-Conductance Calcium-Activated Potassium Channel Activator, CTIBD, and Its Relaxation Effect on Urinary Bladder Smooth Muscle.

The large-conductance calcium-activated potassium channel (BKCa channel) is expressed on various tissues and is involved in smooth muscle relaxation. The channel is highly expressed on urinary bladder smooth muscle cells and regulates the repolarization phase of the spontaneous action potentials that control muscle contraction. To discover novel chemical activators of the BKCa channel, we screened a chemical library containing 8364 chemical compounds using a cell-based fluorescence assay. A chemical compound containing an isoxazolyl benzene skeleton (compound 1) was identified as a potent activator of the BKCa channel and was structurally optimized through a structure-activity relationship study to obtain 4-(4-(4-chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol (CTIBD). When CTIBD was applied to the treated extracellular side of the channel, the conductance-voltage relationship of the channel shifted toward a negative value, and the maximum conductance increased in a concentration-dependent manner. CTIBD altered the gating kinetics of the channel by dramatically slowing channel closing without effecting channel opening. The effects of CTIBD on bladder muscle relaxation and micturition function were tested in rat tissue and in vivo. CTIBD concentration-dependently reduced acetylcholine-induced contraction of urinary bladder smooth muscle strips. In an acetic acid-induced overactive bladder (OAB) model, intraperitoneal injection of 20 mg/kg CTIBD effectively restored frequent voiding contraction and lowered voiding volume without affecting other bladder function parameters. Thus, our results indicate that CTIBD and its derivatives are novel chemical activators of the bladder BKCa channel and potential candidates for OAB therapeutics. SIGNIFICANCE STATEMENT: The novel BKCa channel activator CTIBD was identified and characterized in this study. CTIBD directly activates the BKCa channel and relaxes urinary bladder smooth muscle of rat, so CTIBD can be a potential candidate for overactive bladder therapeutics.

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.

Nitric oxide mediates stretch-induced Ca2+ oscillation in smooth muscle

The stretching of smooth muscle tissue modulates contraction through augmentation of Ca(2+) transients, but the mechanism underlying stretch-induced Ca(2+) transients is still unknown. We found that mechanical stretching and maintenance of mouse urinary bladder smooth muscle strips and single myocytes at 30% and 18% beyond the initial length, respectively, resulted in Ca(2+) oscillations. Experiments indicated that mechanical stretching remarkably increased the production of nitric oxide (NO) as well as the amplitude and duration of muscle contraction. Stretch-induced Ca(2+) oscillations and contractility increases were completely abolished by the NO inhibitor L-NAME or eNOS (also known as NOS3) gene inactivation. Moreover, exposure of eNOS-knockout myocytes to exogenous NO donor induced Ca(2+) oscillations. The stretch-induced Ca(2+) oscillations were greatly inhibited by the selective inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor xestospongin C and partially inhibited by ryanodine. Moreover, the stretch-induced Ca(2+) oscillations were also suppressed by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, but not by the soluble guanylyl cyclase (sGC) inhibitor ODQ. These results suggest that stretching myocyte and maintenance at a certain length results in Ca(2+) oscillations that are NO dependent , and sGC and cGMP independent, and results from the activation of PI3K in smooth muscle.

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

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

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.

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.

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

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

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.

Nerve-released acetylcholine contracts urinary bladder smooth muscle by inducing action potentials independently of IP3-mediated calcium release

Nerve-released ACh is the main stimulus for contraction of urinary bladder smooth muscle (UBSM). Here, the mechanisms by which ACh contracts UBSM are explored by determining Ca(2+) and electrical signals induced by nerve-released ACh. Photolysis of caged inositol 1,4,5-trisphosphate (IP(3)) evoked Ca(2+) release from the sarcoplasmic reticulum. Electrical field stimulation (20 Hz) induced Ca(2+) waves within the smooth muscle that were present only during stimulus application. Ca(2+) waves were blocked by inhibition of muscarinic ACh receptors (mAChRs) with atropine and depletion of sarcoplasmic reticulum Ca(2+) stores with cyclopiazonic acid (CPA), and therefore likely reflect activation of IP(3) receptors (IP(3)Rs). Electrical field stimulation also increased excitability to induce action potentials (APs) that were accompanied by Ca(2+) flashes, reflecting Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCCs) during the action potential. The evoked Ca(2+) flashes and APs occurred as a burst with a lag time of approximately 1.5 s after onset of stimulation. They were not inhibited by blocking IP(3)-mediated Ca(2+) waves, but by blockers of mAChRs (atropine) and VDCCs (diltiazem). Nerve-evoked contractions of UBSM strips were greatly reduced by blocking VDCCs, but not by preventing IP(3)-mediated Ca(2+) signaling with cyclopiazonic acid or inhibition of PLC with U73122. These results indicate that ACh released from nerve varicosities induces IP(3)-mediated Ca(2+) waves during stimulation; but contrary to expectations, these signals do not appear to participate in contraction. In addition, our data provide compelling evidence that UBSM contractions evoked by nerve-released ACh depend on increased excitability and the resultant Ca(2+) entry through VDCCs during APs.

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.

BK channel activation by NS11021 decreases excitability and contractility of urinary bladder smooth muscle

Large-conductance Ca(2+)-activated potassium (BK) channels play an important role in regulating the function and activity of urinary bladder smooth muscle (UBSM), and the loss of BK channel function has been shown to increase UBSM excitability and contractility. However, it is not known whether activation of BK channels has the converse effect of reducing UBSM excitability and contractility. Here, we have sought to investigate this possibility by using the novel BK channel opener NS11021. NS11021 (3 microM) caused an approximately threefold increase in both single BK channel open probability (P(o)) and whole cell BK channel currents. The frequency of spontaneous action potentials in UBSM strips was reduced by NS11021 from a control value of 20.9 + or - 5.9 to 10.9 + or - 3.7 per minute. NS11021 also reduced the force of UBSM spontaneous phasic contractions by approximately 50%, and this force reduction was blocked by pretreatment with the BK channel blocker iberiotoxin. NS11021 (3 microM) had no effect on contractions evoked by nerve stimulation. These findings indicate that activating BK channels reduces the force of UBSM spontaneous phasic contractions, principally through decreasing the frequency of spontaneous action potentials.

Nerve‐evoked purinergic signalling suppresses action potentials, Ca2+ flashes and contractility evoked by muscarinic receptor activation in mouse urinary bladder smooth muscle

Contraction of urinary bladder smooth muscle (UBSM) is caused by the release of ATP and ACh from parasympathetic nerves. Although both purinergic and muscarinic pathways are important to contraction, their relative contributions and signalling mechanisms are not well understood. Here, the contributions of each pathway to urinary bladder contraction and the underlying electrical and Ca(2+) signalling events were examined in UBSM strips from wild type mice and mice deficient in P2X1 receptors (P2X1(-/-)) before and after pharmacological inhibition of purinergic and muscarinic receptors. Electrical field stimulation was used to excite parasympathetic nerves to increase action potentials, Ca(2+) flash frequency, and force. Loss of P2X1 function not only eliminated action potentials and Ca(2+) flashes during stimulation, but it also led to a significant increase in Ca(2+) flashes following stimulation and a corresponding increase in the force transient. Block of muscarinic receptors did not affect action potentials or Ca(2+) flashes during stimulation, but prevented them following stimulation. These findings indicate that nerve excitation leads to rapid engagement of smooth muscle P2X1 receptors to increase action potentials (Ca(2+) flashes) during stimulation, and a delayed increase in excitability in response to muscarinic receptor activation. Together, purinergic and muscarinic stimulation shape the time course of force transients. Furthermore, this study reveals a novel inhibitory effect of P2X1 receptor activation on subsequent increases in muscarinic-driven excitability and force generation.

Signaling Processes for Initiating Smooth Muscle Contraction upon Neural Stimulation

Relationships among biochemical signaling processes involved in Ca2+/calmodulin (CaM)-dependent phosphorylation of smooth muscle myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) were determined. A genetically-encoded biosensor MLCK for measuring Ca(2+)-dependent CaM binding and activation was expressed in smooth muscles of transgenic mice. We performed real-time evaluations of the relationships among [Ca2+](i), MLCK activation, and contraction in urinary bladder smooth muscle strips neurally stimulated for 3 s. Latencies for the onset of [Ca2+](i) and kinase activation were 55 +/- 8 and 65 +/- 6 ms, respectively. Both increased with RLC phosphorylation at 100 ms, whereas force latency was 109 +/- 3 ms. [Ca2+](i), kinase activation, and RLC phosphorylation responses were maximal by 1.2 s, whereas force increased more slowly to a maximal value at 3 s. A delayed temporal response between RLC phosphorylation and force is probably due to mechanical effects associated with elastic elements in the tissue. MLCK activation partially declined at 3 s of stimulation with no change in [Ca2+](i) and also declined more rapidly than [Ca2+](i) during relaxation. The apparent desensitization of MLCK to Ca2+ activation appears to be due to phosphorylation in its calmodulin binding segment. Phosphorylation of two myosin light chain phosphatase regulatory proteins (MYPT1 and CPI-17) or a protein implicated in strengthening membrane adhesion complexes for force transmission (paxillin) did not change during force development. Thus, neural stimulation leads to rapid increases in [Ca2+](i), MLCK activation, and RLC phosphorylation in phasic smooth muscle, showing a tightly coupled Ca2+ signaling complex as an elementary mechanism initiating contraction.

Stromatoxin‐1‐sensitive voltage‐gated K+ channels regulate rat urinary bladder smooth muscle contractility

Recent studies report that stromatoxin‐1 (ScTx1), a 34‐amino acid peptide, selectively inhibits the voltage‐gated K+ (Kv) channel members, Kv2.1, Kv2.2, Kv4.2, and Kv2.1/9.3 heteromultimers with high affinity. The objective of this study was to identify if any voltage‐gated Kv channels sensitive to ScTx1 participate in the regulation of rat urinary bladder smooth muscle (UBSM) contractility. Freshly isolated rat UBSM strips and single UBSM cells were studied using molecular, immunological, and physiological techniques. RT‐PCR experiments on isolated UBSM cells show mRNA expression of Kv2.1, Kv2.2, and Kv9.3 but not Kv4.2 subunits (n=3). Protein expression of Kv2.1 and Kv2.2 was further confirmed in single UBSM cells by applying immunological methods with specific antibodies (n=3). Isometric UBSM tension recordings of isolated UBSM strips showed that ScTx1 (100 nM) increased spontaneous phasic contraction amplitude and muscle force by about 20% without any effect on contraction frequency or muscle tone (n=5). The ScTx1 (100 nM) effect on UBSM contraction amplitude was observed even after the UBSM strips were depolarized with 20 mM K+ (n=3). Our studies indicate that Kv2.1 and Kv2.2 members of the Kv family play a key role in shaping rat UBSM contractility. New drugs targeting specific Kv channel subunits in the UBSM may prove useful for overactive bladder.Supported by NIH DK‐070909 to G.V. Petkov.

Stimulation of β3-adrenoceptors relaxes rat urinary bladder smooth muscle via activation of the large-conductance Ca2+-activated K+ channels

We investigated the role of large-conductance Ca(2+)-activated K(+) (BK) channels in beta3-adrenoceptor (beta3-AR)-induced relaxation in rat urinary bladder smooth muscle (UBSM). BRL 37344, a specific beta3-AR agonist, inhibits spontaneous contractions of isolated UBSM strips. SR59230A, a specific beta3-AR antagonist, and H89, a PKA inhibitor, reduced the inhibitory effect of BRL 37344. Iberiotoxin, a specific BK channel inhibitor, shifts the BRL 37344 concentration response curves for contraction amplitude, net muscle force, and tone to the right. Freshly dispersed UBSM cells and the perforated mode of the patch-clamp technique were used to determine further the role of beta3-AR stimulation by BRL 37344 on BK channel activity. BRL 37344 increased spontaneous, transient, outward BK current (STOC) frequency by 46.0 +/- 20.1%. In whole cell mode at a holding potential of V(h) = 0 mV, the single BK channel amplitude was 5.17 +/- 0.28 pA, whereas in the presence of BRL 37344, it was 5.55 +/- 0.41 pA. The BK channel open probability was also unchanged. In the presence of ryanodine and nifedipine, the current-voltage relationship in response to depolarization steps in the presence and absence of BRL 37344 was identical. In current-clamp mode, BRL 37344 caused membrane potential hyperpolarization from -26.1 +/- 2.1 mV (control) to -29.0 +/- 2.2 mV. The BRL 37344-induced hyperpolarization was eliminated by application of iberiotoxin, tetraethylammonium or ryanodine. The data indicate that stimulation of beta3-AR relaxes rat UBSM by increasing the BK channel STOC frequency, which causes membrane hyperpolarization and thus relaxation.

β-Adrenergic relaxation of mouse urinary bladder smooth muscle in the absence of large-conductance Ca2+-activated K+ channel

In urinary bladder smooth muscle (UBSM), stimulation of beta-adrenergic receptors (beta-ARs) leads to activation of the large-conductance Ca2+-activated K+ (BK) channel currents (Petkov GV and Nelson MT. Am J Physiol Cell Physiol 288: C1255-C1263, 2005). In this study we tested the hypothesis that the BK channel mediates UBSM relaxation in response to beta-AR stimulation using the highly specific BK channel inhibitor iberiotoxin (IBTX) and a BK channel knockout (BK-KO) mouse model in which the gene for the pore-forming subunit was deleted. UBSM strips isolated from wild-type (WT) and BK-KO mice were stimulated with 20 mM K+ or 1 microM carbachol to induce phasic and tonic contractions. BK-KO and WT UBSM strips pretreated with IBTX had increased overall contractility, and UBSM BK-KO cells were depolarized with approximately 12 mV. Isoproterenol, a nonspecific beta-AR agonist, and forskolin, an adenylate cyclase activator, decreased phasic and tonic contractions of WT UBSM strips in a concentration-dependent manner. In the presence of IBTX, the concentration-response curves to isoproterenol and forskolin were shifted to the right in WT UBSM strips. Isoproterenol- and forskolin-mediated relaxations were enhanced in BK-KO UBSM strips, and a leftward shift in the concentration-response curves was observed. The leftward shift was eliminated upon PKA inhibition with H-89, suggesting upregulation of the beta-AR-cAMP pathway in BK-KO mice. These results indicate that the BK channel is a key modulator in beta-AR-mediated relaxation of UBSM and further suggest that alterations in BK channel expression or function could contribute to some pathophysiological conditions such as overactive bladder and urinary incontinence.