Detrusor Smooth Muscle Excitability Research Articles

PTHrP attenuates spontaneous contractions in detrusor smooth muscle of the rat bladder by activating spontaneous transient outward potassium currents.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) cells upon bladder distension attenuates spontaneous phasic contractions (SPCs) in DSM and associated afferent firing to facilitate urine storage. Here, we investigate the mechanisms underlying PTHrP-induced inhibition of SPCs, focusing on large-conductance Ca2+-activated K+ channels (BK channels) that play a central role in stabilizing DSM excitability. Perforated patch-clamp techniques were applied to DSM cells of the rat bladder dispersed using collagenase. Isometric tension changes were recorded from DSM strips, while intracellular Ca2+ dynamics were visualized using Cal520AM-loaded DSM bundles. DSM cells developed spontaneous transient outward potassium currents (STOCs) arising from the opening of BK channels. PTHrP (10nM) increased the frequency of STOCs without affecting their amplitude at a holding potential of - 30mV but not - 40mV. PTHrP enlarged depolarization-induced, BK-mediated outward currents at membrane potentials positive to + 20mV in a manner sensitive to iberiotoxin (100nM), the BK channel blocker. The PTHrP-induced increases in BK currents were also prevented by inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (CPA 10µM), L-type voltage-dependent Ca2+ channel (LVDCC) (nifedipine 3µM) or adenylyl cyclase (SQ22536 100µM). PTHrP had no effect on depolarization-induced LVDCC currents. PTHrP suppressed and slowed SPCs in an iberiotoxin (100nM)-sensitive manner. PTHrP also reduced the number of Ca2+ spikes during each burst of spontaneous Ca2+ transients. In conclusion, PTHrP accelerates STOCs discharge presumably by facilitating SR Ca2+ release which prematurely terminates Ca2+ transient bursts resulting in the attenuation of SPCs.

Detrusor Smooth Muscle KV7 Channels: Emerging New Regulators of Urinary Bladder Function.

Relaxation and contraction of the urinary bladder smooth muscle, also known as the detrusor smooth muscle (DSM), facilitate the micturition cycle. DSM contractility depends on cell excitability, which is established by the synchronized activity of multiple diverse ion channels. K+ channels, the largest family of channels, control DSM excitability by maintaining the resting membrane potential and shaping the action potentials that cause the phasic contractions. Among the members of the voltage-gated K+ (KV) channel superfamily, KV type 7 (KV7) channels — KV7.1–KV7.5 members encoded by KCNQ1–KCNQ5 genes — have been recently identified as functional regulators in various cell types including vascular, cardiac, and neuronal cells. Their regulatory roles in DSM, however, are just now emerging and remain to be elucidated. To address this gap, our research group has initiated the systematic investigation of human DSM KV7 channels in collaboration with clinical urologists. In this comprehensive review, we summarize the current understanding of DSM Kv7 channels and highlight recent discoveries in the field. We describe KV7 channel expression profiles at the mRNA and protein levels, and further elaborate on functional effects of KV7 channel selective modulators on DSM excitability, contractility, and intracellular Ca2+ dynamics in animal species along with in vivo studies and the limited data on human DSM. Within each topic, we highlight the main observations, current gaps in knowledge, and most pressing questions and concepts in need of resolution. We emphasize the lack of systematic studies on human DSM KV7 channels that are now actively ongoing in our laboratory.

Functional heterogeneity of PDGFRα (+) cells in spontaneously active urogenital tissues.

As PDGFRα (+) cells appear not to suppress the excitability of detrusor smooth muscle by generating SK3-dependent hyperpolarising as proposed in the gastrointestinal tract, we further explored the functional roles of PDGFRα (+) cells in regulating the spontaneous activity of urogenital tissues. Using PDGFRα-eGFP mice, intracellular Ca2+ signaling in PDGFRα (+) cells of the bladder lamina propria, renal pelvis, and seminal vesicle were visualized using Cal-590 fluorescence. The distribution and SK3 expression of PDGFRα (+) cells were also examined by immunohistochemistry. In the bladder lamina propria, SK3 (-) PDGFRα (+) cells exhibited spontaneous Ca2+ transients and responded to stimulation of P2Y1 purinoceptors with MRS2365 (100 nM) or adenosine diphosphate (ADP) (100 μM) by developing Ca2+ transients. In the proximal renal pelvis, PDGFRα (+) cells were distributed in the mucosal, muscular and serosal layers but did not express SK3 immunoreactivity. PDGFRα (+) cells in the musculature resembling atypical smooth muscle cells generated spontaneous Ca2+ transients that were partially suppressed upon P2Y1-stimulation, while vigorously responding to human angiotensin II (100 nM). In the seminal vesicle, PDGFRα (+) cells in the musculature but not mucosa expressed SK3 immunoreactivity. In the mucosa, the P2Y1 stimulation evoked Ca2+ transients in both PDGFRα (+) cells and PDGFRα (-) cells. PDGFRα (+) cells in spontaneously active urogenital tissues display heterogeneity in terms of their SK3 expression and P2Y1-induced Ca2+ responses. Muscular PDGFRα (+) cells in the renal pelvis and mucosal PDGFRα (+) cells in the seminal vesicle may generate depolarizing signals to drive smooth muscle cells.

In Silico Pharmacological Assessment of Mibefradil in Single Detrusor Smooth Muscle Cell towards Understanding Urinary Bladder Overactivity

OBJECTIVESEnhanced spontaneous contraction of the detrusor smooth muscle (DSM) cells, which is also known as DSM instability is associated with overactive bladder (OAB), a pathophysiological syndrome affecting millions of individuals socially. Due to adverse side effects of conventional anticholinergic drugs, researchers are focusing on novel drug compounds with high specificity. Understanding the drug effects with respect to various ion channels offers additional possibilities for safety pharmacological assessment. Here our overarching objective was to utilize computational approaches to simulate the effects of the T‐type Ca2+ channel blocker Mibefradil on the DSM cell action potential (AP) and subsequent DSM cell excitability.METHODSThe DSM cell model is described as an equivalent electrical circuit consisting of a membrane capacitance connected in parallel with a number of variable conductances representing two voltage‐gated Ca2+ (T ‐ type and L‐ type) channels, two voltage‐gated potassium (Kv, KCNQ) channels, three calcium‐dependent potassium (BK, IK and SK) channels, an ATP‐dependent potassium channel and an inward rectifying cation channel. A drug model for Mibefradil was simulated by multiplying the maximal conductance of T‐type Ca2+ channel with a scaling factor between 0 and 1 to mimic the drug concentration.KEY RESULTSIn this model, all ionic conductances were tuned to set the resting membrane potential (RMP) at − 50 mV. A synaptic input for 15 ms was injected to evoke the AP. The maximum conductance value of the T‐type Ca2+ channel (g−CaT) was set to 0.0006 S/cm2. Adding Mibefradil by 50% of its control value reduced the peak amplitude of AP and inward current. However, the addition of Mibefradil by 100% resulted in no AP and zero inward current. Representative APs are shown in Fig 1 (A) with proper legends. The T‐ type Ca2+ conductance was varied by up to ± 50 % of the control value in discrete steps to study the RMP sensitivity analysis. The normalized changes in RMP of the AP is shown in Fig 1(B). Note that RMP varies at most by up to + 10% and − 15%, indicating T‐ type Ca2+ conductance dependent DSM cell excitability. These results (Fig 1A and 1B) show that T‐type Ca2+ channels play an important role in generating AP and deactivation of T‐type Ca2+ channels decrease the excitability of DSM cell.CONCLUSIONS AND IMPLICATIONSWe investigated the ability of the Mibefradil to modulate DSM cell’s AP using our computational model which provides a virtual electrophysiological workbench. This in silico assessment showed that inhibition of T‐type Ca2+ channels hyperpolarized the RMP, eliminated the AP and reduced DSM cell excitability. This study suggests that a compound, such as Mibefradil may form a part of new pharmacological strategy in the treatment of OAB.Support or Funding InformationThis work is supported in part by Department of Biotechnology (DBT), India (grant number BT/PR12973/MED/122/47/2016).(A) Effects of Mibefradil on synaptic input based AP (B) RMP Sensitivity analysis with varying T‐ type Ca2+ conductance.Figure 1

TRPM4 channel inhibitors 9-phenanthrol and glibenclamide differentially decrease guinea pig detrusor smooth muscle whole-cell cation currents and phasic contractions.

Nonselective cation channels, consistent with transient receptor potential melastatin-4 (TRPM4), regulate detrusor smooth muscle (DSM) function. TRPM4 channels can exist as homomers or assemble with sulfonylurea receptors (SURs) as complexes. We evaluated contributions of TRPM4/SUR-TRPM4 channels to DSM excitability and contractility by examining the effects of TRPM4/SUR-TRPM4 channel modulators 9-phenanthrol, glibenclamide, and diazoxide on freshly-isolated guinea pig DSM cells (amphotericin-B perforated patch-clamp electrophysiology) and mucosa-free DSM strips (isometric tension recordings). In DSM cells, complete removal of extracellular Na+ decreased voltage-step-induced cation (non-K+ selective) currents. At high positive membrane potentials, 9-phenanthrol at 100 μM attenuated voltage step-induced currents more effectively than at 30 μM, revealing concentration-dependent, voltage-sensitive inhibition. In comparison to 9-phenanthrol, glibenclamide (100 μM) displayed lower inhibition of cation currents. In the presence of glibenclamide (100 μM), 9-phenanthrol (100 μM) further decreased the currents. The SUR-TRPM4 complex activator diazoxide (100-300 μM) weakly inhibited the currents. 9-Phenanthrol, but not glibenclamide or diazoxide, increased cell capacitance (a cell surface area indicator). In contractility studies, glibenclamide displayed lower potencies than 9-phenanthrol attenuating spontaneous and 20 mM KCl-induced DSM phasic contractions. While both compounds showed similar maximum inhibitions on DSM spontaneous phasic contractions, glibenclamide was generally less efficacious on 20 mM KCl-induced phasic contractions. In summary, the observed differential effects of 9-phenanthrol and glibenclamide on DSM excitability and contractility support unique mechanisms for the two compounds. The data suggest that SUR-TRPM4 complexes do not contribute to DSM function. This study advances our understanding of pharmacological effects of glibenclamide and 9-phenanthrol on DSM cell cation currents.

Extracellular pH and intracellular phosphatidylinositol 4,5-bisphosphate control Cl- currents in guinea pig detrusor smooth muscle cells.

Cl- channels serve as key regulators of excitability and contractility in vascular, intestinal, and airway smooth muscle cells. We recently reported a Cl- conductance in detrusor smooth muscle (DSM) cells. Here, we used the whole cell patch-clamp technique to further characterize biophysical properties and physiological regulators of the Cl- current in freshly isolated guinea pig DSM cells. The Cl- current demonstrated outward rectification arising from voltage-dependent gating of Cl- channels rather than the Cl- transmembrane gradient. An exposure of DSM cells to hypotonic extracellular solution (Δ 165 mOsm challenge) did not increase the Cl- current providing strong evidence that volume-regulated anion channels do not contribute to the Cl- current in DSM cells. The Cl- current was monotonically dependent on extracellular pH, larger and lower in magnitude at acidic (5.0) and basic pH (8.5) values, respectively. Additionally, intracellularly applied phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] analog [PI(4,5)P2-diC8] increased the average Cl- current density by approximately threefold in a voltage-independent manner. The magnitude of the DSM whole cell Cl- current did not depend on the cell surface area (cell capacitance) regardless of the presence or absence of PI(4,5)P2-diC8, an intriguing finding that underscores the complex nature of Cl- channel expression and function in DSM cells. Removal of both extracellular Ca2+ and Mg2+ did not affect the DSM whole cell Cl- current, whereas Gd3+ (1 mM) potentiated the current. Collectively, our recent and present findings strongly suggest that Cl- channels are critical regulators of DSM excitability and are regulated by extracellular pH, Gd3+, and PI(4,5)P2.

A computational model of large conductance voltage and calcium activated potassium channels: implications for calcium dynamics and electrophysiology in detrusor smooth muscle cells.

The large conductance voltage and calcium activated potassium (BK) channels play a crucial role in regulating the excitability of detrusor smooth muscle, which lines the wall of the urinary bladder. These channels have been widely characterized in terms of their molecular structure, pharmacology and electrophysiology. They control the repolarising and hyperpolarising phases of the action potential, thereby regulating the firing frequency and contraction profiles of the smooth muscle. Several groups have reported varied profiles of BK currents and I-V curves under similar experimental conditions. However, no single computational model has been able to reconcile these apparent discrepancies. In view of the channels' physiological importance, it is imperative to understand their mechanistic underpinnings so that a realistic model can be created. This paper presents a computational model of the BK channel, based on the Hodgkin-Huxley formalism, constructed by utilising three activation processes - membrane potential, calcium inflow from voltage-gated calcium channels on the membrane and calcium released from the ryanodine receptors present on the sarcoplasmic reticulum. In our model, we attribute the discrepant profiles to the underlying cytosolic calcium received by the channel during its activation. The model enables us to make heuristic predictions regarding the nature of the sub-membrane calcium dynamics underlying the BK channel's activation. We have employed the model to reproduce various physiological characteristics of the channel and found the simulated responses to be in accordance with the experimental findings. Additionally, we have used the model to investigate the role of this channel in electrophysiological signals, such as the action potential and spontaneous transient hyperpolarisations. Furthermore, the clinical effects of BK channel openers, mallotoxin and NS19504, were simulated for the detrusor smooth muscle cells. Our findings support the proposed application of these drugs for amelioration of the condition of overactive bladder. We thus propose a physiologically realistic BK channel model which can be integrated with other biophysical mechanisms such as ion channels, pumps and exchangers to further elucidate its micro-domain interaction with the intracellular calcium environment.

Compound‐dependent Effects of TRPM4 Channel Modulators on Guinea Pig Detrusor Smooth Muscle Excitability and Contractility

Guinea pig detrusor smooth muscle (DSM) cells express transient receptor potential melastatin 4 (TRPM4) channels, proposed to regulate DSM excitability and contractility1. Supporting evidence with the TRPM4 channel inhibitor 9‐phenanthrol showed reduced guinea pig DSM phasic contractions, and induced DSM cell hyperpolarization and decreased voltage‐step evoked cation currents1. Recently, a concern has been raised about selectivity of 9‐phenanthrol2–4. Here, we used whole‐cell amphotericin‐B perforated and inside‐out single‐channel patch‐clamp techniques, and isometric DSM tension recordings to address the hypothesis that TRPM4 channels regulate DSM excitation‐contraction coupling by examining the novel selective TRPM4 channel inhibitor 4‐chloro‐2‐[2‐(2‐chloro‐phenoxy)‐acetylamino]‐benzoic acid (CBA, compound 5 in reference5), along with non‐selective TRPM4 channel modulators: flufenamic acid (FFA)5 and BTP2 (or YM‐58483)6, a blocker and an activator, respectively. DSM cell voltage‐step induced cation currents were not increased when held at +26 or +6 mV in‐between voltage steps in comparison to the standard −74 mV (n=7), inconsistent with presence of TRPM4 currents6. Separate applications of CBA (30 μM) or BTP2 (10 μM) in DSM cells did not change the voltage‐step evoked currents (n=5–6). In contrast, FFA (100 μM) caused an increase at positive voltages, 1.4‐fold at +106 mV (p<0.05, n=7). In the same DSM cells, 9‐phenanthrol (100 μM) added following washout of FFA or in the presence of either CBA or BTP2 reduced the currents, 42–57% at +106 mV (p<0.05, n=4–7). 9‐Phenanthrol but not CBA, FFA, or BTP2 altered cell capacitance, 1.1‐fold increase (p<0.05). Excised single‐channel recordings revealed a non‐TRPM4 channel cation conductance of ~15 pS that was blocked by 9‐phenanthrol (30 μM). CBA, added cumulatively up to 100 μM, showed either no or very weak effects (maximum inhibitions: 8–38%) on spontaneous and 20 mM KCl‐induced phasic contraction parameters (n=10–13). For 300 μM CBA, inhibitions were higher (up to 68%) on all contraction parameters except for the duration of KCl‐induced contractions, which increased 2‐fold (n=7–9). 9‐Phenanthrol examined in parallel showed robust reductions in DSM phasic contraction parameters (IC50 values: 1.1–21 μM and maximum inhibitions: 35–86%, n=11–20). In summary, 9‐phenanthrol ‐ in contrast to other tested TRPM4 channel modulators: CBA, FFA, or BTP2 ‐effectively reduced DSM cell voltage‐step induced cation currents and DSM strip contractions. Our results reveal differential effects of TRPM4 channel modulators on guinea pig DSM excitability and contractility. Future studies are needed to determine the underlying mechanisms and to elucidate the function of TRPM4 in the urinary bladder. Interpretation of the results with 9‐phenanthrol requires caution especially related to its perceived TRPM4 channel selectivity.Support or Funding InformationNIH DK106964 to Georgi V. PetkovThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Properties of single-channel and whole cell Cl- currents in guinea pig detrusor smooth muscle cells.

Multiple types of Cl- channels regulate smooth muscle excitability and contractility in vascular, gastrointestinal, and airway smooth muscle cells. However, little is known about Cl- channels in detrusor smooth muscle (DSM) cells. Here, we used inside-out single channel and whole cell patch-clamp recordings for detailed biophysical and pharmacological characterizations of Cl- channels in freshly isolated guinea pig DSM cells. The recorded single Cl- channels displayed unique gating with multiple subconductive states, a fully opened single-channel conductance of 164 pS, and a reversal potential of -41.5 mV, which is close to the ECl of -65 mV, confirming preferential permeability to Cl-. The Cl- channel demonstrated strong voltage dependence of activation (half-maximum of mean open probability, V0.5, ~-20 mV) and robust prolonged openings at depolarizing voltages. The channel displayed similar gating when exposed intracellularly to solutions containing Ca2+-free or 1 mM Ca2+. In whole cell patch-clamp recordings, macroscopic current demonstrated outward rectification, inhibitions by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid, and insensitivity to chlorotoxin. The outward current was reversibly reduced by 94% replacement of extracellular Cl- with I-, Br-, or methanesulfonate (MsO-), resulting in anionic permeability sequence: Cl->Br->I->MsO-. While intracellular Ca2+ levels (0, 300 nM, and 1 mM) did not affect the amplitude of Cl- current and outward rectification, high Ca2+ slowed voltage-step current activation at depolarizing voltages. In conclusion, our data reveal for the first time the presence of a Ca2+-independent DIDS and niflumic acid-sensitive, voltage-dependent Cl- channel in the plasma membrane of DSM cells. This channel may be a key regulator of DSM excitability.

KV7 Channel Pharmacological Activation by the Novel Activator ML213: Role for Heteromeric KV7.4/KV7.5 Channels in Guinea Pig Detrusor Smooth Muscle Function.

Voltage-gated KV7 channels (KV7.1 to KV7.5) are important regulators of the cell membrane potential in detrusor smooth muscle (DSM) of the urinary bladder. This study sought to further the current knowledge of KV7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca2+ imaging, amphotericin-B perforated patch-clamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound N-(2,4,6-trimethylphenyl)-bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of KV7.2, KV7.4, and KV7.5 channels, to examine their physiologic roles in guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 µM) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the KV7 channel inhibitor XE991 (10 µM). ML213 (0.1-30 µM) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 µM) decreased global intracellular Ca2+ concentrations in Fura-2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 µM) caused an increase in the amplitude of whole-cell KV7 currents. Further, in current-clamp mode of the perforated patch clamp, ML213 hyperpolarized DSM cell membrane potential in a manner reversible by washout or XE991 (10 µM), consistent with ML213 activation of KV7 channel currents. Preapplication of XE991 (10 µM) not only depolarized the DSM cells, but also blocked ML213-induced hyperpolarization, confirming ML213 selectivity for KV7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric KV7.4/KV7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive KV7.4- and KV7.5-containing channels are essential regulators of DSM excitability and contractility.

Nongenomic modulation of the large conductance voltage- and Ca2+-activated K+ channels by estrogen: A novel regulatory mechanism in human detrusor smooth muscle.

Estrogens have an important role in regulating detrusor smooth muscle (DSM) function. However, the underlying molecular and cellular mechanisms by which estrogens control human DSM excitability and contractility are not well known. Here, we used human DSM specimens from open bladder surgeries on 27 patients to elucidate the mechanism by which 17β‐estradiol regulates large conductance voltage‐ and Ca2+‐activated K+ (BK) channels, the most prominent K+ channels in human DSM. We employed single BK channel recordings on inside‐out excised membrane patches, perforated whole‐cell patch‐clamp on freshly isolated DSM cells, and isometric tension recordings on DSM‐isolated strips to investigate the mechanism by which 17β‐estradiol activates BK channels. 17β‐Estradiol (100 nmol/L) rapidly increased depolarization‐induced whole‐cell K+ currents in DSM cells. The 17β‐estradiol stimulatory effects on whole‐cell BK currents were completely abolished by the selective BK channel inhibitor paxilline (1 μmol/L), clearly indicating that 17β‐estradiol specifically activates BK channels. 17β‐Estradiol also increased the frequency of ryanodine receptor‐mediated transient BK currents. Single BK channel recordings showed that 17β‐estradiol (100 nmol/L) significantly increased the BK channel open probability of inside‐out excised membrane patches, revealing that 17β‐estradiol activates BK channels directly. 17β‐Estradiol reduced spontaneous phasic contractions of human DSM‐isolated strips in a concentration‐dependent manner (100 nmol/L‐1 μmol/L), and this effect was blocked by paxilline (1 μmol/L). 17β‐Estradiol (100 nmol/L) also reduced nerve‐evoked contractions of human DSM‐isolated strips. Collectively, our results reveal that 17β‐estradiol plays a critical role in regulating human DSM function through a direct nongenomic activation of BK channels.

AB317. SPR-44 Pharmacological activation of individual KCNQ channel subtypes in detrusor smooth muscle represents a promising novel approach for overactive bladder treatment

ObjectiveOur recent studies have demonstrated voltage-gated KCNQ channels (KCNQ1-KCNQ5) as key regulators of detrusor smooth muscle (DSM) function. Despite emerging developments, the physiological role of individual KCNQ channel subtypes remains less clear. Here, we utilized the novel compound ML-213, a potent activator of KCNQ2, KCNQ4, and KCNQ5 channels, to elucidate their physiological roles in guinea pig DSM function.MethodsUsing isometric DSM tension recordings, Ca2+ imaging, and amphotericin-B perforated patch-clamp electrophysiology, we elucidated the role of ML-213-sensitive KCNQ channels in regulating DSM excitability and contractility.ResultsML-213 concentration-dependently (100 nM–30 µM) inhibited spontaneous phasic, pharmacologically-induced, and nerve-evoked contractions in DSM isolated strips. ML-213 (10 µM) decreased the global intracellular Ca2+ concentrations in DSM isolated strips, effects blocked by the L-type voltage-gated Ca2+ (CaV) channel inhibitor nifedipine (1 µM) and the KCNQ1-KCNQ5 channel inhibitor XE991 (10 µM). These data suggest that ML-213 decreases the global intracellular Ca2+ concentration by inhibiting L-type CaV channels through an indirect mechanism downstream from KCNQ channel activation. In addition, ML-213 hyperpolarized the cell membrane potential and inhibited spontaneous action potentials in DSM cells, effects reversible by washout. We next aimed to examine the effects of ML-213 on whole cell KCNQ currents. To isolate KCNQ currents, the bath solution contained the large conductance voltage- and Ca2+-activated K+ channel inhibitor paxilline (1 µM) and gadolinium chloride (GdCl3, 50 µM), which blocks L-type CaV channels and non-selective cation channels. Under these experimental conditions, ML-213 (10 µM) enhanced whole cell KCNQ currents. These findings suggest that the modulation of K+ transport through ML-213-sensitive KCNQ channels underlies ML-213-induced cell membrane hyperpolarization to decrease the global intracellular Ca2+ concentration and DSM contractility.ConclusionsThese data using the novel compound ML-213, suggest that KCNQ2-, KCNQ4-, and KCNQ5-containing channels are essential regulators of the excitability, intracellular Ca2+ concentration, and contractility of DSM by virtue of their control of the membrane potential. Moreover, these new findings provide a foundational basis for future investigations on KCNQ channel functional roles in human DSM excitability and contractility to confirm their potential as novel therapeutic targets for overactive bladder.Source of FundingNIH grant R01-DK106964 to GV Petkov and F31-DK104528 to A Provence.

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

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

Effects of K+ channel openers on spontaneous action potentials in detrusor smooth muscle of the guinea-pig urinary bladder

The modulation of spontaneous excitability in detrusor smooth muscle (DSM) upon the pharmacological activation of different populations of K+ channels was investigated. Effects of distinct K+ channel openers on spontaneous action potentials in DSM of the guinea-pig bladder were examined using intracellular microelectrode techniques. NS1619 (10μM), a large conductance Ca2+-activated K+ (BK) channel opener, transiently increased action potential frequency and then prevented their generation without hyperpolarizing the membrane in a manner sensitive to iberiotoxin (IbTX, 100nM). A higher concentration of NS1619 (30μM) hyperpolarized the membrane and abolished action potential firing. NS309 (10μM) and SKA31 (100μM), small conductance Ca2+-activated K+ (SK) channel openers, dramatically increased the duration of the after-hyperpolarization and then abolished action potential firing in an apamin (100nM)-sensitive manner. Flupirtine (10μM), a Kv7 channel opener, inhibited action potential firing without hyperpolarizing the membrane in a manner sensitive to XE991 (10μM), a Kv7 channel blocker. BRL37344 (10μM), a β3-adrenceptor agonist, or rolipram (10nM), a phosphodiesterase 4 inhibitor, also inhibited action potential firing. A higher concentration of rolipram (100nM) hyperpolarized the DSM and abolished the action potentials. IbTX (100nM) prevented the rolipram-induced blockade of action potentials but not the hyperpolarization. BK and Kv7 channels appear to predominantly contribute to the stabilization of DSM excitability. Spare SK channels could be pharmacologically activated to suppress DSM excitability. BK channels appear to be involved in the cyclic AMP-induced inhibition of action potentials but not the membrane hyperpolarization.

BK channel regulation by phosphodiesterase type 1: a novel signaling pathway controlling human detrusor smooth muscle function.

Large-conductance Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) function. We aimed to investigate phosphodiesterase type 1 (PDE1) interactions with BK channels in human DSM to determine the mechanism by which PDE1 regulates human urinary bladder physiology. A combined electrophysiological, functional, and pharmacological approach was applied using human DSM 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 DSM cells in combination with the selective PDE1 inhibitor, 8-methoxymethyl-3-isobutyl-1-methylxanthine (8MM-IBMX). Isometric DSM tension recordings were used to measure spontaneous phasic and electrical field stimulation-induced contractions in human DSM isolated strips. Selective pharmacological inhibition of PDE1 with 8MM-IBMX (10 μM) increased TBKC activity in human DSM 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 DSM cell membrane potential, an effect blocked by PKA inhibition. 8MM-IBMX significantly decreased spontaneous phasic and nerve-evoked contractions of human DSM isolated strips. The results reveal a novel mechanism that pharmacological inhibition of PDE1 attenuates human DSM 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 DSM. Inhibition of PDE1 can be a novel therapeutic approach for the treatment of overactive bladder associated with detrusor overactivity.

Novel regulatory mechanism in human urinary bladder: central role of transient receptor potential melastatin 4 channels in detrusor smooth muscle function.

Transient receptor potential melastatin 4 (TRPM4) channels are Ca(2+)-activated nonselective cation channels that have been recently identified as regulators of detrusor smooth muscle (DSM) function in rodents. However, their expression and function in human DSM remain unexplored. We provide insights into the functional role of TRPM4 channels in human DSM under physiological conditions. We used a multidisciplinary experimental approach, including RT-PCR, Western blotting, immunohistochemistry and immunocytochemistry, patch-clamp electrophysiology, and functional studies of DSM contractility. DSM samples were obtained from patients without preoperative overactive bladder symptoms. RT-PCR detected mRNA transcripts for TRPM4 channels in human DSM whole tissue and freshly isolated single cells. Western blotting and immunohistochemistry with confocal microscopy revealed TRPM4 protein expression in human DSM. Immunocytochemistry further detected TRPM4 protein expression in DSM single cells. Patch-clamp experiments showed that 9-phenanthrol, a selective TRPM4 channel inhibitor, significantly decreased the transient inward cation currents and voltage step-induced whole cell currents in freshly isolated human DSM cells. In current-clamp mode, 9-phenanthrol hyperpolarized the human DSM cell membrane potential. Furthermore, 9-phenanthrol attenuated the spontaneous phasic, carbachol-induced and nerve-evoked contractions in human DSM isolated strips. Significant species-related differences in TRPM4 channel activity between human, rat, and guinea pig DSM were revealed, suggesting a more prominent physiological role for the TRPM4 channel in the regulation of DSM function in humans than in rodents. In conclusion, TRPM4 channels regulate human DSM excitability and contractility and are critical determinants of human urinary bladder function. Thus, TRPM4 channels could represent promising novel targets for the pharmacological or genetic control of overactive bladder.

Regulation of Guinea Pig Detrusor Smooth Muscle Excitability by 17β-Estradiol: The Role of the Large Conductance Voltage- and Ca2+-Activated K+ Channels.

Estrogen replacement therapies have been suggested to be beneficial in alleviating symptoms of overactive bladder. However, the precise regulatory mechanisms of estrogen in urinary bladder smooth muscle (UBSM) at the cellular level remain unknown. Large conductance voltage- and Ca2+-activated K+ (BK) channels, which are key regulators of UBSM function, are suggested to be non-genomic targets of estrogens. This study provides an electrophysiological investigation into the role of UBSM BK channels as direct targets for 17β-estradiol, the principle estrogen in human circulation. Single BK channel recordings on inside-out excised membrane patches and perforated whole cell patch-clamp were applied in combination with the BK channel selective inhibitor paxilline to elucidate the mechanism of regulation of BK channel activity by 17β-estradiol in freshly-isolated guinea pig UBSM cells. 17β-Estradiol (100 nM) significantly increased the amplitude of depolarization-induced whole cell steady-state BK currents and the frequency of spontaneous transient BK currents in freshly-isolated UBSM cells. The increase in whole cell BK currents by 17β-estradiol was eliminated upon blocking BK channels with paxilline. 17β-Estradiol (100 nM) significantly increased (~3-fold) the single BK channel open probability, indicating direct 17β-estradiol-BK channel interactions. 17β-Estradiol (100 nM) caused a significant hyperpolarization of the membrane potential of UBSM cells, and this hyperpolarization was reversed by blocking the BK channels with paxilline. 17β-Estradiol (100 nM) had no effects on L-type voltage-gated Ca2+ channel currents recorded under perforated patch-clamp conditions. This study reveals a new regulatory mechanism in the urinary bladder whereby BK channels are directly activated by 17β-estradiol to reduce UBSM cell excitability.

The Novel KV7.2/KV7.3 Channel Opener ICA-069673 Reveals Subtype-Specific Functional Roles in Guinea Pig Detrusor Smooth Muscle Excitability and Contractility.

The physiologic roles of voltage-gated KV7 channel subtypes (KV7.1-KV7.5) in detrusor smooth muscle (DSM) are poorly understood. Here, we sought to elucidate the functional roles of KV7.2/KV7.3 channels in guinea pig DSM excitability and contractility using the novel KV7.2/KV7.3 channel activator ICA-069673 [N-(2-chloro-5-pyrimidinyl)-3,4-difluorobenzamide]. We employed a multilevel experimental approach using Western blot analysis, immunocytochemistry, isometric DSM tension recordings, fluorescence Ca(2+) imaging, and perforated whole-cell patch-clamp electrophysiology. Western blot experiments revealed the protein expression of KV7.2 and KV7.3 channel subunits in DSM tissue. In isolated DSM cells, immunocytochemistry with confocal microscopy further confirmed protein expression for KV7.2 and KV7.3 channel subunits, where they localize within the vicinity of the cell membrane. ICA-069673 inhibited spontaneous phasic, pharmacologically induced, and nerve-evoked contractions in DSM isolated strips in a concentration-dependent manner. The inhibitory effects of ICA-069673 on DSM spontaneous phasic and tonic contractions were abolished in the presence of the KV7 channel inhibitor XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride]. Under conditions of elevated extracellular K(+) (60 mM), the effects of ICA-069673 on DSM tonic contractions were significantly attenuated. ICA-069673 decreased the global intracellular Ca(2+) concentration in DSM cells, an effect blocked by the L-type Ca(2+) channel inhibitor nifedipine. ICA-069673 hyperpolarized the membrane potential and inhibited spontaneous action potentials of isolated DSM cells, effects that were blocked in the presence of XE991. In conclusion, using the novel KV7.2/KV7.3 channel activator ICA-069673, this study provides strong evidence for a critical role for the KV7.2- and KV7.3-containing channels in DSM function at both cellular and tissue levels.

Prostaglandin E2 excitatory effects on guinea pig urinary bladder smooth muscle: A novel regulatory mechanism mediated by large-conductance voltage- and Ca2+-activated K+ channels

Prostaglandin E2 (PGE2) is an essential signaling molecule involved in the regulation of detrusor smooth muscle (DSM) function. However, the underlying regulatory mechanism by which PGE2 augments DSM cell excitability and contractility is not well understood. Here, we investigated whether PGE2 inhibits the large conductance voltage- and Ca2+-activated K+ (BK) channels in guinea pig DSM, thereby increasing DSM excitability and contractility. We used a multidisciplinary experimental approach including amphotericin–B perforated patch-clamp electrophysiology and live-cell Ca2+ imaging in native freshly-isolated DSM cells, isometric tension recordings of intact DSM strips, and pharmacological tools to investigate BK channel regulation by PGE2 in guinea pig DSM. PGE2 increased the spontaneous phasic contractions of isolated DSM strips in a concentration-dependent manner (10nM–10µM). BK channel inhibition with paxilline (1µM) attenuated the PGE2-induced DSM phasic contractions, suggesting that BK channels are involved in the mechanism of PGE2-induced DSM contractions. PGE2 (10µM) increased the intracellular Ca2+ levels in freshly-isolated DSM cells. PGE2 (10µM) also caused an inhibition of the amplitude and frequency of spontaneous transient BK currents in DSM cells. Moreover, PGE2 (10µM) did not affect the amplitude of whole cell steady-state BK currents in DSM cells. Our findings provide strong experimental evidence that PGE2 leads to an inhibition of the spontaneous transient BK currents, elevation of intracellular Ca2+ levels in freshly-isolated DSM cells, and augmentation of DSM phasic contractions. Thus, we have revealed a novel mechanism that BK channels mediate PGE2-induced contractions in guinea pig DSM.

Functional link between muscarinic receptors and large-conductance Ca2+ -activated K+ channels in freshly isolated human detrusor smooth muscle cells.

Activation of muscarinic acetylcholine receptors (mAChRs) constitutes the primary mechanism for enhancing excitability and contractility of human detrusor smooth muscle (DSM). Since the large-conductance Ca(2+)-activated K(+) (KCa1.1) channels are key regulators of human DSM function, we investigated whether mAChR activation increases human DSM excitability by inhibiting KCa1.1 channels. We used the mAChR agonist, carbachol, to determine the changes in KCa1.1 channel activity upon mAChR activation in freshly isolated human DSM cells obtained from open bladder surgeries using the perforated whole cell and single KCa1.1 channel patch-clamp recordings. Human DSM cells were collected from 29 patients (23 males and 6 females, average age of 65.9 ± 1.5 years). Carbachol inhibited the amplitude and frequency of KCa1.1 channel-mediated spontaneous transient outward currents and spontaneous transient hyperpolarizations, which are triggered by the release of Ca(2+) from ryanodine receptors. Carbachol also caused membrane potential depolarization, which was not observed in the presence of iberiotoxin, a KCa1.1 channel inhibitor, indicating the critical role of the KCa1.1 channels. The potential direct carbachol effects on KCa1.1 channels were examined under conditions of removing the major cellular Ca(2+) sources for KCa1.1 channel activation with pharmacological inhibitors (thapsigargin, ryanodine, and nifedipine). In the presence of these inhibitors, carbachol did not affect the single KCa1.1 channel open probability and mean KCa1.1 channel conductance (cell-attached configuration) or depolarization-induced whole cell steady-state KCa1.1 currents. The data support the concept that mAChR activation triggers indirect functional KCa1.1 channel inhibition mediated by intracellular Ca(2+), thus increasing the excitability in human DSM cells.