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.

Age-dependent decrease in TRPM4 channel expression but not trafficking alters urinary bladder smooth muscle contractility.

During development, maturation, or aging, the expression and function of urinary bladder smooth muscle (UBSM) ion channels can change, thus affecting micturition. Increasing evidence supports a novel role of transient receptor potential melastatin-4 (TRPM4) channels in UBSM physiology. However, it remains unknown whether the functional expression of these key regulatory channels fluctuates in UBSM over different life stages. Here, we examined TRPM4 channel protein expression (Western blot) and the effects of TRPM4 channel inhibitors, 9-phenanthrol and glibenclamide, on phasic contractions of UBSM isolated strips obtained from juvenile (UBSM-J, 5-9 weeks old) and adult (UBSM-A, 6-18 months old) male guinea pigs. Compared to UBSM-J, UBSM-A displayed a 50-70% reduction in total TRPM4 protein expression, while the surface-to-intracellular expression ratio (channel trafficking) remained the same in both age groups. Consistent with the reduced total TRPM4 protein expression in UBSM-A, 9-phenanthrol showed lower potencies and/or maximum efficacies in UBSM-A than UBSM-J for inhibiting amplitude and muscle force of spontaneous and 20 mM KCl-induced phasic contractions. Compared to 9-phenanthrol, glibenclamide also attenuated both spontaneous and KCl-induced contractions, but with less pronounced differential effects in UBSM-A and UBSM-J. In both age groups, regardless of the overall reduced total TRPM4 protein expression in UBSM-A, cell surface TRPM4 protein expression (~80%) predominated over its intracellular fraction (~20%), revealing preserved channel trafficking mechanisms toward the cell membrane. Collectively, this study reports novel findings illuminating a fundamental physiological role for TRPM4 channels in UBSM function that fluctuates with age.

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.

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.

Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents.

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, fresh, and isolated DSM cells presents an important technical challenge whose achievement provides optimal cells for subsequent functional and molecular studies. The method developed and elaborated herein, successfully used by our group for over a decade, describes dissection of human urinary bladder specimens obtained from open bladder surgeries followed by an enzymatic two-step treatment of DSM pieces and mechanical trituration to obtain freshly isolated DSM cells. The initial step involves dissection to separate the DSM layer (also known as muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa) and the adjacent connective, vascular, and adipose tissues present. The DSM is then cut into pieces (2-3 mm x 4-6 mm) in nominal Ca2+-containing dissection/digestion solution (DS). DSM 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 DS containing enzyme-free bovine serum and trituration with a fire-polished pipette, the pieces release single DSM cells. Freshly isolated DSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations of ion channels. Specifically, we show that the TRPM4 channel blocker 9-phenanthrol reduces voltage-step evoked cation currents recorded with the amphotericin-B perforated patch-clamp approach. DSM 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. The main advantage of utilizing single DSM cells is that the observations made relate directly to single cell characteristics revealed. Studies of freshly isolated human DSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable in the urinary bladder and will continue as a gold standard in elucidating DSM cellular properties and regulatory mechanisms.

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.

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 17ß-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 17ß-estradiol activates BK channels. 17ß-Estradiol (100 nmol/L) rapidly increased depolarization-induced whole-cell K+ currents in DSM cells. The 17ß-estradiol stimulatory effects on whole-cell BK currents were completely abolished by the selective BK channel inhibitor paxilline (1 µmol/L), clearly indicating that 17ß-estradiol specifically activates BK channels. 17ß-Estradiol also increased the frequency of ryanodine receptor-mediated transient BK currents. Single BK channel recordings showed that 17ß-estradiol (100 nmol/L) significantly increased the BK channel open probability of inside-out excised membrane patches, revealing that 17ß-estradiol activates BK channels directly. 17ß-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). 17ß-Estradiol (100 nmol/L) also reduced nerve-evoked contractions of human DSM-isolated strips. Collectively, our results reveal that 17ß-estradiol plays a critical role in regulating human DSM function through a direct nongenomic activation of BK channels.

Regulation of transient receptor potential melastatin 4 channel by sarcoplasmic reticulum inositol trisphosphate receptors: Role in human detrusor smooth muscle function.

We recently reported key physiologic roles for Ca2+-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca2+-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca2+, inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the sarcoplasmic reticulum represents a potential Ca2+ source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IP3Rs and TRPM4 channels. With in situ proximity ligation assay (PLA) and perforated patch-clamp electrophysiology, we tested the hypothesis that TRPM4 channels are tightly associated with the IP3Rs and are activated by IP3R-mediated Ca2+ release in human DSM. With in situ PLA, we demonstrated co-localization of the TRPM4 channels and IP3Rs in human DSM cells. As the TRPM4 channels and IP3Rs must be located within close apposition to functionally interact, these findings support the concept of a potential Ca2+-mediated TRPM4-IP3R regulatory mechanism. 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 DSM cells, blocking the IP3Rs with the selective IP3R inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IP3Rs have a key role in mediating the Ca2+-dependent activation of TRPM4 channels in human DSM. The study provides novel insight into the molecular and cellular mechanisms regulating TRPM4 channels by revealing that TRPM4 channels and IP3Rs are spatially and functionally coupled in human DSM.

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.

Constitutively active PKA regulates neuronal acetylcholine release and contractility of guinea pig urinary bladder smooth muscle.

Autonomic and somatic motor neurons that innervate the urinary bladder and urethra control the highly coordinated functions of the lower urinary tract, the storage, and the emptying of urine. ACh is the primary excitatory neurotransmitter in the bladder. Here, we aimed to determine whether PKA regulates neuronal ACh release and related nerve-evoked detrusor smooth muscle (DSM) contractions in the guinea pig urinary bladder. Isometric DSM tension recordings were used to measure spontaneous phasic and electrical field stimulation (EFS)- and carbachol-induced DSM contractions with a combination of pharmacological tools. The colorimetric method was used to measure ACh released by the parasympathetic nerves in DSM isolated strips. The pharmacological inhibition of PKA with H-89 (10 µM) increased the spontaneous phasic contractions, whereas it attenuated the EFS-induced DSM contractions. Intriguingly, H-89 (10 µM) attenuated the (primary) cholinergic component, whereas it simultaneously increased the (secondary) purinergic component of the nerve-evoked contractions in DSM isolated strips. The acetylcholinesterase inhibitor, eserine (10 µM), increased EFS-induced DSM contractions, and the subsequent addition of H-89 attenuated the contractions. H-89 (10 µM) significantly increased DSM phasic contractions induced by the cholinergic agonist carbachol. The inhibition of PKA decreased the neuronal release of ACh in DSM tissues. This study revealed that PKA-mediated signaling pathways differentially regulate nerve-evoked and spontaneous phasic contractions of guinea pig DSM. Constitutively active PKA in the bladder nerves controls synaptic ACh release, thus regulating the nerve-evoked DSM contractions, whereas PKA in DSM cells controls the spontaneous phasic contractility.

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.

Do we need to know more about the effects of hormones on lower urinary tract dysfunction? ICI-RS 2014.

This review article reflects the presentations and subsequent discussions during a think tank at the 5th International Consultation on Incontinence Research Society's annual meeting, held in Bristol, UK (September 22-24, 2014). It reviews the current state of knowledge on the role of hormones in lower urinary tract dysfunction (LUTD) and overactive bladder (OAB) and in particular: highlights some specific basic research findings from discussion participants; reviews future research topics; and discusses potential new therapeutic opportunities for LUTD and OAB. The role of the large conductance voltage- and Ca(2+) -activated K(+) (BK) channels, as novel therapeutic targets for OAB was discussed, in particular as recent studies on human detrusor smooth muscle suggest that estradiol exerts a direct non-genomic activation of the BK channels. Recent developments on the roles of sex hormones on diuresis, as well as the roles of melatonin and vitamin D on LUTD were also discussed. It was concluded that further basic science and translational studies are needed to better understand hormonal regulatory mechanisms of the lower urinary tract and the implications for novel treatment options for LUTD and OAB.

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 17ß-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 17ß-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 17ß-estradiol in freshly-isolated guinea pig UBSM cells. 17ß-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 17ß-estradiol was eliminated upon blocking BK channels with paxilline. 17ß-Estradiol (100 nM) significantly increased (~3-fold) the single BK channel open probability, indicating direct 17ß-estradiol-BK channel interactions. 17ß-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. 17ß-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 17ß-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.

Novel mechanism of hydrogen sulfide-induced guinea pig urinary bladder smooth muscle contraction: role of BK channels and cholinergic neurotransmission.

Hydrogen sulfide (H2S) is a key signaling molecule regulating important physiological processes, including smooth muscle function. However, the mechanisms underlying H2S-induced detrusor smooth muscle (DSM) contractions are not well understood. This study investigates the cellular and tissue mechanisms by which H2S regulates DSM contractility, excitatory neurotransmission, and large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels in freshly isolated guinea pig DSM. We used a multidisciplinary experimental approach including isometric DSM tension recordings, colorimetric ACh measurement, Ca(2+) imaging, and patch-clamp electrophysiology. In isolated DSM strips, the novel slow release H2S donor, P-(4-methoxyphenyl)-p-4-morpholinylphosphinodithioic acid morpholine salt (GYY4137), significantly increased the spontaneous phasic and nerve-evoked DSM contractions. The blockade of neuronal voltage-gated Na(+) channels or muscarinic ACh receptors with tetrodotoxin or atropine, respectively, reduced the stimulatory effect of GYY4137 on DSM contractility. GYY4137 increased ACh release from bladder nerves, which was inhibited upon blockade of L-type voltage-gated Ca(2+) channels with nifedipine. Furthermore, GYY4137 increased the amplitude of the Ca(2+) transients and basal Ca(2+) levels in isolated DSM strips. GYY4137 reduced the DSM relaxation induced by the BK channel opener, NS11021. In freshly isolated DSM cells, GYY4137 decreased the amplitude and frequency of transient BK currents recorded in a perforated whole cell configuration and reduced the single BK channel open probability measured in excised inside-out patches. GYY4137 inhibited spontaneous transient hyperpolarizations and depolarized the DSM cell membrane potential. Our results reveal the novel findings that H2S increases spontaneous phasic and nerve-evoked DSM contractions by activating ACh release from bladder nerves in combination with a direct inhibition of DSM BK channels.

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.