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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.

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.

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.

New Findings What is the central question of this study? The aim was to investigate the roles of extracellular chloride in electrical slow waves and resting membrane potential of mouse jejunal smooth muscle by replacing chloride with the impermeant anions gluconate and isethionate. What is the main finding and its importance? The main finding was that in smooth muscle cells, the resting Cl− conductance is low, whereas transmembrane Cl− movement in interstitial cells of Cajal (ICCs) is a major contributor to the shape of electrical slow waves. Furthermore, the data confirm that ICCs set the smooth muscle membrane potential and that altering Cl− homeostasis in ICCs can alter the smooth muscle membrane potential. Intracellular Cl− homeostasis is regulated by anion-permeable channels and transporters and contributes to excitability of many cell types, including smooth muscle and interstitial cells of Cajal (ICCs). Our aims were to investigate the effects on electrical activity in mouse jejunal muscle strips of replacing extracellular Cl− (Cl−o) with the impermeant anions gluconate and isethionate. On reducing Cl−o, effects were observed on electrical slow waves, with small effects on smooth muscle membrane voltage (Em). Restoration of Cl− hyperpolarized smooth muscle Em proportional to the change in Cl−o concentration. Replacement of 90% of Cl−o with gluconate reversibly abolished slow waves in five of nine preparations. Slow waves were maintained in isethionate. Gluconate and isethionate substitution had similar concentration-dependent effects on peak amplitude, frequency, width at half peak amplitude, rise time and decay time of residual slow waves. Gluconate reduced free ionized Ca2+ in Krebs solutions to 0.13 mm. In Krebs solutions containing normal Cl− and 0.13 mm free Ca2+, slow wave frequency was lower, width at half peak amplitude was smaller, and decay time was faster. The transient hyperpolarization following restoration of Cl−o was not observed in W/Wv mice, which lack pacemaker ICCs in the small intestine. We conclude that in smooth muscle cells, the resting Cl− conductance is low, whereas transmembrane Cl− movement in ICCs plays a major role in generation or propagation of slow waves. Furthermore, these data support a role for ICCs in setting smooth muscle Em and that altering Cl− homeostasis in ICCs can alter smooth muscle Em.

Myenteric plexus interstitial cells of Cajal (ICC-MY) in the small intestine are Kit+ electrical pacemakers that express the Ano1/TMEM16A Ca2+-activated Cl– channel, whose functions in the gastrointestinal tract remain incompletely understood. In this study, an inducible Cre-LoxP-based approach was used to advance the understanding of Ano1 in ICC-MY of adult mouse small intestine. KitCreERT2/+;Ano1Fl/Fl mice were treated with tamoxifen or vehicle, and small intestines (mucosa free) were examined. Quantitative RT-PCR demonstrated ~50% reduction in Ano1 mRNA in intestines of conditional knockouts (cKOs) compared with vehicle-treated controls. Whole mount immunohistochemistry showed a mosaic/patchy pattern loss of Ano1 protein in ICC networks. Ca2+ transients in ICC-MY network of cKOs displayed reduced duration compared with highly synchronized controls and showed synchronized and desynchronized profiles. When matched, the rank order for Ano1 expression in Ca2+ signal imaged fields of view was as follows: vehicle controls>>>cKO(synchronized)>cKO(desynchronized). Maintenance of Ca2+ transients’ synchronicity despite high loss of Ano1 indicates a large functional reserve of Ano1 in the ICC-MY network. Slow waves in cKOs displayed reduced duration and increased inter-slow-wave interval and occurred in regular- and irregular-amplitude oscillating patterns. The latter activity suggested ongoing interaction by independent interacting oscillators. Lack of slow waves and depolarization, previously reported for neonatal constitutive knockouts, were also seen. In summary, Ano1 in adults regulates gastrointestinal function by determining Ca2+ transients and electrical activity depending on the level of Ano1 expression. Partial Ano1 loss results in Ca2+ transients and slow waves displaying reduced duration, while complete and widespread absence of Ano1 in ICC-MY causes lack of slow wave and desynchronized Ca2+ transients. NEW & NOTEWORTHY The Ca2+-activated Cl− channel, Ano1, in interstitial cells of Cajal (ICC) is necessary for normal gastrointestinal motility. We knocked out Ano1 to varying degrees in ICC of adult mice. Partial knockout of Ano1 shortened the widths of electrical slow waves and Ca2+ transients in myenteric ICC but Ca2+ transient synchronicity was preserved. Near-complete knockout was necessary for transient desynchronization and loss of slow waves, indicating a large functional reserve of Ano1 in ICC. View this article's corresponding video summary at https://youtu.be/cyPtDP0KLY4 .

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.

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.

Transient receptor potential melastatin 4 (TRPM4) channels are Ca2+-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.

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 E(Cl) of −65 mV, confirming preferential permeability to Cl(−). The Cl(−)channel demonstrated strong voltage dependence of activation (half-maximum of mean open probability, V(0.5), ~−20 mV) and robust prolonged openings at depolarizing voltages. The channel displayed similar gating when exposed intracellularly to solutions containing Ca(2+)-free or 1 mM Ca(2+). 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 Ca(2+) levels (0, 300 nM, and 1 mM) did not affect the amplitude of Cl(−) current and outward rectification, high Ca(2+) slowed voltage-step current activation at depolarizing voltages. In conclusion, our data reveal for the first time the presence of a Ca(2+)-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.

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.

Intracellular Cl− homeostasis is regulated by anion-permeable channels and transporters and contributes to excitability of many cell types including smooth muscle and interstitial cells of Cajal (ICC). Our aims were to investigate the effects on electrical activity in mouse jejunal muscle strips, of substituting extracellular Cl− (Cl−o) with the impermeant anions, gluconate and isethionate. On reducing Cl−o, effects were observed on electrical slow waves with small effects on smooth muscle membrane voltage (Em). Restoration of Cl− hyperpolarized smooth muscle Em proportional to the change in Cl−o concentration. Replacement of 90% of Cl−o with gluconate reversibly abolished slow waves in 5/9 preparations. Slow waves were maintained in isethionate. Gluconate and isethionate substitution had similar concentration dependent effects on peak amplitude, frequency, width at half peak amplitude, rise time and decay time of residual slow waves. Gluconate reduced free ionized Ca2+ in Krebs solutions to 0.13 mM. In Krebs solutions containing normal Cl− and 0.13 mM free Ca2+, slow wave frequency was lower, width at half peak amplitude was smaller and decay time was faster. The transient hyperpolarization following restoration of Cl−o was not observed in W/Wv mice, which lack pacemaker ICC in the small intestine. We conclude that in smooth muscle cells, the resting Cl− conductance is low, whereas transmembrane Cl− movement in ICC plays a major role in generation or propagation of slow waves. Furthermore, these data support a role for ICC in setting smooth muscle Em and that altering Cl− homeostasis in ICC can alter smooth muscle Em.

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.

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

Members of the transient receptor potential (TRP) channel superfamily, including the Ca2+-activated monovalent cation-selective TRP melastatin 4 (TRPM4) channel, have been recently identified in the urinary bladder. However, their expression and function at the level of detrusor smooth muscle (DSM) remain largely unexplored. In this study, for the first time we investigated the role of TRPM4 channels in guinea pig DSM excitation-contraction coupling using a multidisciplinary approach encompassing protein detection, electrophysiology, live-cell Ca2+ imaging, DSM contractility, and 9-phenanthrol, a recently characterized selective inhibitor of the TRPM4 channel. Western blot and immunocytochemistry experiments demonstrated the expression of the TRPM4 channel in whole DSM tissue and freshly isolated DSM cells with specific localization on the plasma membrane. Perforated whole cell patch-clamp recordings and real-time Ca2+ imaging experiments with fura 2-AM, both using freshly isolated DSM cells, revealed that 9-phenanthrol (30 μM) significantly reduced the cation current and decreased intracellular Ca2+ levels. 9-Phenanthrol (0.1–30 μM) significantly inhibited spontaneous, 0.1 μM carbachol-induced, 20 mM KCl-induced, and nerve-evoked contractions in guinea pig DSM-isolated strips with IC50 values of 1–7 μM and 70–80% maximum inhibition. 9-Phenanthrol also reduced nerve-evoked contraction amplitude induced by continuous repetitive electrical field stimulation of 10-Hz frequency and shifted the frequency-response curve (0.5–50 Hz) relative to the control. Collectively, our data demonstrate the novel finding that TRPM4 channels are expressed in guinea pig DSM and reveal their critical role in the regulation of guinea pig DSM excitation-contraction coupling.

The Ca2+-activated Cl− channel, Ano1, in interstitial cells of Cajal (ICC) is necessary for normal gastrointestinal motility. We knocked out Ano1 to varying degrees in ICC of adult mice. Partial knockout of Ano1 shortened the widths of electrical slow waves and Ca2+ transients in myenteric ICC but Ca2+ transient synchronicity was preserved. Near-complete knockout was necessary for transient desynchronization and loss of slow waves, indicating a large functional reserve of Ano1 in ICC.

α7 nicotinic acetylcholine receptors (nAChRs) are characterized by relatively low ACh sensitivity, rapid activation, and fast desensitization kinetics. ACh/agonist evoked currents at the α7 nAChR are transient, and, typically, calcium flux responses are difficult to detect using conventional fluorometric assay techniques. One approach to study interactions of agonists with the α7 nAChR is by utilizing positive allosteric modulators (PAMs). In this study, we demonstrate that inclusion of type II PAMs such as PNU-120596, but not type I, can enable detection of endogenous α7 nAChR-mediated calcium responses in human neuroblastoma (IMR-32) cells. Using this approach, we characterized the pharmacological profile of nicotine, epibatidine, choline, and other nAChR agonists such as PNU-282987, SSR-180711, GTS-21, OH-GTS21, tropisetron, NS6784, and A-582941. The rank order potency of agonists well correlated with α7 nAChR binding affinities measured in brain membranes. Inhibition of calcium response by methyllycaconitine in the presence of increasing concentrations of PNU-282987 or PNU-120596 revealed that the IC(50) value of methyllycaconitine was sensitive to varying concentrations of the agonist, but not that of the PAM. This format demonstrated the feasibility of this approach for high-throughput screening to identify small molecule, PAMs, which were further confirmed in electrophysiological assays of human α7 nAChR expressed in oocytes.

Genistein and 5-hydroxyindole (5-HI) potentiate the α7 nicotinic acetylcholine receptor current by primarily increasing peak amplitude, a property of type I α7 positive allosteric modulation. In this study, the effects of these two compounds were investigated at two different α7/5-HT(3) chimeras (chimera 1, comprising of extracellular α7 N-terminus fused to the remainder of 5-HT(3A), and chimera 2 containing an additional α7 encoded M2-M3 loop), and wild-type α7 and 5-HT(3A) receptors. Agonist-evoked responses, examined by expression of the chimeras in Xenopus laevis oocytes or HEK-293 cells, revealed that currents decayed slower and compounds {rank order: N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987)~2-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5-phenyl-1,3,4-oxadiazole (NS6784)>acetylcholine>choline} were more potent in chimera 2 than chimera 1 or α7 receptors. In chimera 2, genistein and 5-HI potentiated agonist-evoked responses (EC(50): 4-5 μM for genistein and 300-500 μM for 5-HI) and at higher concentrations evoked current directly consistent with ago-allosteric modulation. At chimera 1 and 5-HT(3A) receptors, neither compound directly evoked any current and 5-HI, only at chimera 1, was able to potentiate agonist-evoked responses. Genistein and 5-HI did not inhibit the binding of the α7 agonist [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1] heptane ([(3)H]A-585539) to rat brain or chimera 2. In summary, this study supports the role of the M2-M3 loop being critical for the positive allosteric effect of genistein, but not 5-HI, and in agonist-evoked response fine-tuning. The identification of distinct α7 receptor modulatory sites offers unique opportunities for developing CNS therapeutics and understanding its pharmacology.

Alpha3-containing (alpha 3*) and alpha 7 nicotinic acetylcholine receptors (nAChRs) are expressed in human IMR-32 neuroblastoma cells and implicated in Ca(2+) signaling. In this study, we investigated the intracellular Ca(2+) transient generation evoked by selective activation of alpha 3* (agonist potency rank order: epibatidinevareniclinenicotine approximately cytisine) and alpha 7 (rank order in the presence of alpha 7 positive allosteric modulator or PAM: A-795723NS6784 approximately PNU-282987) using, respectively, varenicline and NS6784 (+alpha 7 PAM) by Ca(2+) imaging. Effects of inhibitors of nAChRs (MLA and mecamylamine), ER Ca(2+) ATPase pump (CPA and thapsigargin), Ca(2+)-induced Ca(2+) release (ryanodine and dantrolene), Ca(2+) channels (nitrendipine, diltiazem, and Cd(2+)), and removal of extracellular Ca(2+) were examined. alpha 7 PAMs, when tested in the presence of NS6784, were more active when added first, followed by the agonist, than in the reverse order. Removal of extracellular Ca(2+) - but not CPA, thapsigargin, ryanodine, dantrolene, nitrendipine, diltiazem, or Cd(2+) - diminished the alpha 7 agonist-evoked Ca(2+) transients. In contrast, only diltiazem and nitrendipine and removal of extracellular Ca(2+) inhibited the alpha 3*-mediated Ca(2+) transients. The differential effect of diltiazem and nitrendipine versus Cd(2+) was due to direct inhibition of alpha 3* nAChRs as revealed by Ca(2+) imaging in HEK-293 cells expressing human alpha 3 beta 4 nAChRs and patch clamp in IMR-32 cells. In summary, this study provides evidence that alpha 3* and alpha 7 nAChR agonist-evoked global Ca(2+) transient generation in IMR-32 cells does not primarily involve voltage-dependent Ca(2+) channels, intracellular Ca(2+) stores, or Ca(2+)-induced Ca(2+) release. These mechanisms may, however, be still involved in other forms of nAChR-mediated Ca(2+) signaling.

Enhancement of alpha7 nicotinic acetylcholine receptor (nAChR) activity is considered a therapeutic approach for ameliorating cognitive deficits present in Alzheimer's disease and schizophrenia. In this study, we describe the in vitro profile of a novel selective alpha7 nAChR agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). ABT-107 displayed high affinity binding to alpha7 nAChRs [rat or human cortex, [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539), K(i) = 0.2-0.6 nM or [(3)H]methyllycaconitine (MLA), 7 nM] that was at least 100-fold selective versus non-alpha7 nAChRs and other receptors. Functionally, ABT-107 did not evoke detectible currents in Xenopus oocytes expressing human or nonhuman alpha3beta4, chimeric (alpha6/alpha3)beta4, or 5-HT(3A) receptors, and weak or negligible Ca(2+) responses in human neuroblastoma IMR-32 cells (alpha3* function) and human alpha4beta2 and alpha4beta4 nAChRs expressed in human embryonic kidney 293 cells. ABT-107 potently evoked human and rat alpha7 nAChR current responses in oocytes (EC(50), 50-90 nM total charge, approximately 80% normalized to acetylcholine) that were enhanced by the positive allosteric modulator (PAM) 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744). In rat hippocampus, ABT-107 alone evoked alpha7-like currents, which were inhibited by the alpha7 antagonist MLA. In dentate gyrus granule cells, ABT-107 enhanced spontaneous inhibitory postsynaptic current activity when coapplied with A-867744. In the presence of an alpha7 PAM [A-867744 or N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596)], the addition of ABT-107 elicited MLA-sensitive alpha7 nAChR-mediated Ca(2+) signals in IMR-32 cells and rat cortical cultures and enhanced extracellular signal-regulated kinase phosphorylation in differentiated PC-12 cells. ABT-107 was also effective in protecting rat cortical cultures against glutamate-induced toxicity. In summary, ABT-107 is a selective high affinity alpha7 nAChR agonist suitable for characterizing the roles of this subtype in pharmacological studies.

Background and purpose: Several agonists of the α7 nicotinic acetylcholine receptor (nAChR) have been developed for treatment of cognitive deficits. However, agonist efficacy in vivo is difficult to reconcile with rapid α7 nAChR desensitization in vitro; and furthermore, the correlation between in vitro receptor efficacy and in vivo behavioural efficacy is not well delineated. The possibility that agonists of this receptor actually function in vivo as inhibitors via desensitization has not been finally resolved. Experimental approach: Two structurally related α7 nAChR agonists were characterized and used to assess the degree of efficacy required in a behavioural paradigm. Key results: NS6784 activated human and rat α7 nAChR with EC50s of 0.72 and 0.88 µM, and apparent efficacies of 77 and 97% respectively. NS6740, in contrast, displayed little efficacy at α7 nAChR (

alpha4beta2 neuronal nicotinic receptors (nAChRs) can exist in high and low sensitivity states possibly due to distinct stoichiometries during subunit assembly: (alpha4)(2)(beta2)(3) pentamer (high sensitivity, HS) and (alpha4)(3)(beta2)(2) pentamer (low sensitivity, LS). To determine if there is a linkage between HS or LS states and receptor-mediated responses in brain, we profiled several clinically studied alpha4beta2* nAChR agonists for the displacement of radioligand binding to alpha4beta2 [(3)H]-cytisine sites in rat brain membranes, effects on stimulation of [(3)H]-dopamine release from slices of rat prefrontal cortex and striatum, and activation of HS and LS human alpha4beta2 nAChRs expressed in Xenopus laevis oocytes. Binding affinities (pK(i)) and potency (pEC(50)) values for [(3)H]-dopamine release closely correlated with a rank order: varenicline(-)-nicotineAZD3480dianicline congruent with ABT-089. Further, a good correlation was observed between [(3)H]-dopamine release and HS alpha4beta2 pEC(50) values, but not between [(3)H]-dopamine release and LS alpha4beta2. The relative efficacies of the agonists ranged from full to partial agonists. Varenicline behaved as a partial agonist in stimulating [(3)H]-dopamine release and activating both HS and LS alpha4beta2 nAChRs expressed in oocytes. Conversely, ABT-089, AZD3480 and dianicline exhibited little efficacy at LS alpha4beta2 (5%), were more effective at HS alpha4beta2 nAChRs, and in stimulating cortical and striatal [(3)H]-dopamine releaseor=30%. In the presence of alpha-conotoxin MII to block alpha6beta2* nAChRs, the alpha4beta2* alpha-conotoxin-insensitive [(3)H]-dopamine release stimulated by these ligands correlates well with their interactions at HS, but not LS. In summary, this study provides support for HS alpha4beta2* nAChR involvement in neurotransmitter release.

The discovery of a series of pyrrole-sulfonamides as positive allosteric modulators (PAM) of alpha7 nAChRs is described. Optimization of this series led to the identification of 19 (A-867744), a novel type II PAM with good potency and selectivity. Compound 19 showed acceptable pharmacokinetic profile across species and brain levels sufficient to modulate alpha7 nAChRs. In a rodent model of sensory gating, 19 normalized gating deficits. These results suggest that 19 represents a novel class of molecules capable of allosteric modulation of the alpha7 nAChRs.

Targeting alpha7 neuronal acetylcholine receptors (nAChRs) with selective agonists and positive allosteric modulators (PAMs) is considered a therapeutic approach for managing cognitive deficits in schizophrenia and Alzheimer's disease. In this study, we describe a novel type II alpha7 PAM, 4-(5-(4-chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide (A-867744), that exhibits a unique pharmacological profile. In oocytes expressing alpha7 nAChRs, A-867744 potentiated acetylcholine (ACh)-evoked currents, with an EC(50) value of approximately 1 microM. At highest concentrations of A-867744 tested, ACh-evoked currents were essentially nondecaying. At lower concentrations, no evidence of a distinct secondary component was evident in contrast to 4-naphthalen-1-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonic acid amide (TQS), another type II alpha7 PAM. In the presence of A-867744, ACh concentration responses were potentiated by increases in potency, Hill slope, and maximal efficacy. When examined in rat hippocampus CA1 stratum radiatum interneurons or dentate gyrus granule cells, A-867744 (10 microM) increased choline-evoked alpha7 currents and recovery from inhibition/desensitization, and enhanced spontaneous inhibitory postsynaptic current activity. A-867744, like other alpha7 PAMs tested [1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)urea (NS1738), TQS, and 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596)], did not displace the binding of [(3)H]methyllycaconitine to rat cortex alpha7(*) nAChRs. However, unlike these PAMs, A-867744 displaced the binding of the agonist [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539) in rat cortex, with a K(i) value of 23 nM. A-867744 neither increased agonist-evoked responses nor displaced the binding of [(3)H]A-585539 in an alpha7/5-hydroxytryptamine(3) (alpha7/5-HT(3)) chimera, suggesting an interaction distinct from the alpha7 N terminus or M2-3 loop. In addition, A-867744 failed to potentiate responses mediated by 5-HT(3A) or alpha3beta4 and alpha4beta2 nAChRs. In summary, this study identifies a novel and selective alpha7 PAM showing activity at recombinant and native alpha7 nAChRs exhibiting a unique pharmacological interaction with the receptor.

Background and purpose: The α7 nicotinic acetylcholine receptor (nAChR) has attracted considerable interest as a target for cognitive enhancement in schizophrenia and Alzheimer's Disease. However, most recently described α7 agonists are derived from the quinuclidine structural class. Alternatively, the present study identifies tilorone as a novel α7-selective agonist and characterizes analogues developed from this lead. Experimental approach: Activity and selectivity were determined from rat brain α7 and α4β2 nAChR binding, recombinant nAChR activation, and native α7 nAChR mediated stimulation of ERK1/2 phosphorylation in PC12 cells. Key results: Tilorone bound α7 nAChR (IC50 110 nM) with high selectivity relative to α4β2 (IC50 70 000 nM), activated human α7 nAChR with an EC50 value of 2.5 μM and maximal response of 67% relative to acetylcholine, and showed little agonist effect at human α3β4 or α4β2 nAChRs. However, the rat α7 nAChR maximal response was only 34%. Lead optimization led to 2-(5-methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-xanthen-9-one (A-844606) with improved binding (α7 IC50 11 nM, α4β2 IC50>30 000 nM) and activity at both human and rat α7 nAChR (EC50s 1.4 and 2.2 μM and apparent efficacies 61 and 63%, respectively). These compounds also activated native α7 nAChR, stimulating ERK1/2 phosphorylation in PC12 cells. Conclusions and implications: Tilorone, known as an interferon inducer, is a selective α7 nAChR agonist, suggesting utility of the fluorenone pharmacophore for the development of α7 nAChR selective agonists. Whether α7 stimulation mediates interferon induction, or whether interferon induction may influence the potential anti-inflammatory properties of α7 nAChR agonists remains to be elucidated. British Journal of Pharmacology (2008) 153, 1054–1061; doi:10.1038/sj.bjp.0707649; published online 24 December 2007

Among the diverse sets of nicotinic acetylcholine receptors (nAChRs), the alpha7 subtype is highly expressed in the hippocampus and cortex and is thought to play important roles in a variety of cognitive processes. In this review, we describe the properties of a novel biaryl diamine alpha7 nAChR agonist, A-582941. A-582941 was found to exhibit high-affinity binding and partial agonism at alpha7 nAChRs, with acceptable pharmacokinetic properties and excellent distribution to the central nervous system (CNS). In vitro and in vivo studies indicated that A-582941 activates signaling pathways known to be involved in cognitive function such as ERK1/2 and CREB phosphorylation. A-582941 enhanced cognitive performance in behavioral models that capture domains of working memory, short-term recognition memory, memory consolidation, and sensory gating deficit. A-582941 exhibited a benign secondary pharmacodynamic and tolerability profile as assessed in a battery of assays of cardiovascular, gastrointestinal, and CNS function. The studies summarized in this review collectively provide preclinical validation that alpha7 nAChR agonism offers a mechanism with potential to improve cognitive deficits associated with various neurodegenerative and psychiatric disorders.

The α7 nicotinic acetylcholine receptor (nAChR) plays an important role in cognitive processes and may represent a drug target for treating cognitive deficits in neurodegenerative and psychiatric disorders. In the present study, we used a novel α7 nAChR-selective agonist, 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) to interrogate cognitive efficacy, as well as examine potential cellular mechanisms of cognition. Exhibiting high affinity to native rat (Ki= 10.8 nm) and human (Ki= 16.7 nm) α7 nAChRs, A-582941 enhanced cognitive performance in behavioral assays including the monkey delayed matching-to-sample, rat social recognition, and mouse inhibitory avoidance models that capture domains of working memory, short-term recognition memory, and long-term memory consolidation, respectively. In addition, A-582941 normalized sensory gating deficits induced by the α7 nAChR antagonist methyllycaconitine in rats, and in DBA/2 mice that exhibit a natural sensory gating deficit. Examination of signaling pathways known to be involved in cognitive function revealed that α7 nAChR agonism increased extracellular-signal regulated kinase 1/2 (ERK1/2) phosphorylation in PC12 cells. Furthermore, increases in ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation were observed in mouse cingulate cortex and/or hippocampus after acute A-582941 administration producing plasma concentrations in the range of α7 binding affinities and behavioral efficacious doses. The MEK inhibitor SL327 completely blocked α7 agonist-evoked ERK1/2 phosphorylation. Our results demonstrate that α7 nAChR agonism can lead to broad-spectrum efficacy in animal models at doses that enhance ERK1/2 and CREB phosphorylation/activation and may represent a mechanism that offers potential to improve cognitive deficits associated with neurodegenerative and psychiatric diseases, such as Alzheimer's disease and schizophrenia.

Augmentation of nicotinic alpha7 receptor function is considered to be a potential therapeutic strategy aimed at ameliorating cognitive and mnemonic dysfunction in relation to debilitating pathological conditions, such as Alzheimer's disease and schizophrenia. In the present report, a novel positive allosteric modulator of the alpha7 nicotinic acetylcholine receptor (nAChR), 1-(5-chloro-2-hydroxy-phenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)-urea (NS1738), is described. NS1738 was unable to displace or affect radioligand binding to the agonist binding site of nicotinic receptors, and it was devoid of effect when applied alone in electrophysiological paradigms. However, when applied in the presence of acetylcholine (ACh), NS1738 produced a marked increase in the current flowing through alpha7 nAChRs, as determined in both oocyte electrophysiology and patch-clamp recordings from mammalian cells. NS1738 acted by increasing the peak amplitude of ACh-evoked currents at all concentrations; thus, it increased the maximal efficacy of ACh. Oocyte experiments indicated an increase in ACh potency as well. NS1738 had only marginal effects on the desensitization kinetics of alpha7 nAChRs, as determined from patch-clamp studies of both transfected cells and cultured hippocampal neurons. NS1738 was modestly brain-penetrant, and it was demonstrated to counteract a (-)-scopolamine-induced deficit in acquisition of a water-maze learning task in rats. Moreover, NS1738 improved performance in the rat social recognition test to the same extent as (-)-nicotine, demonstrating that NS1738 is capable of producing cognitive enhancement in vivo. These data support the notion that alpha7 nAChR allosteric modulation may constitute a novel pharmacological principle for the treatment of cognitive dysfunction.

We elucidated the physiological role of KV7.2/KV7.3 channels in guinea pig urinary bladder smooth muscle (UBSM) in vitro using the novel KV7.2/KV7.3 channel opener ICA-069673 and a multidisciplinar...

Calcium activated K(+) channels (K(Ca) channels) are found in a variety of smooth muscle tissues, the most characterized of which are the large conductance K(Ca) channels (BK(Ca) or maxi-K(+) channels). Recent medicinal chemistry efforts have identified novel BK(Ca) openers including 2-amino-5-(2-fluoro-phenyl)-4-methyl-1H-pyrrole-3-carbonitrile (NS-8), BMS-204352 and its analog 3-(5-chloro-2-hydroxy-phenyl)-3-hydroxy-6-trifluoromethyl-1,3-dihydro-indol-2-one (compound 1), and 5,7-dichloro-4-(5-chloro-2-hydroxy-phenyl)-3-hydroxy-1H-quinolin-2-one (compound 2). Although these compounds are effective BK(Ca) openers as shown by electrophysiological methods, little is known about their effects on smooth muscle contractility. In this study, the responsiveness of structurally diverse BK(Ca) openers-NS-8, compounds 1 and 2 and the well characterized nonselective NS-1619-was assessed using segments of endothelium denuded rat aorta, rat and guinea pig detrusor precontracted with extracellular K(+), and Landrace pig detrusor stimulated by electrical field. In all preparations, the compounds tested inhibited or completely abolished contractions with similar potencies (-logIC(50) values: 3.8 to 5.1). In rat aorta, in the presence of 80 mM K(+), the compounds significantly shifted the concentration-response curve to the right compared with those obtained in 30 mM K(+). These data are consistent with K(+) channel (BK(Ca) channel) activation as the underlying mechanism of relaxation by compounds that share the electrophysiological property of BK(Ca) current activation. The similar potencies at detrusor and vascular smooth muscle suggest that the achievement of smooth muscle selectivity in vitro with the representative compounds examined in this study may prove to be a challenge when targeting BK(Ca) channels for smooth muscle indications such as overactive bladder.

Although ATP-sensitive K+ channels continue to be explored for their therapeutic potential, developments in high-affinity radioligands to investigate native and recombinant KATP channels have been less forthcoming. This study reports the identification and pharmacological characterization of a novel iodinated 1,4-dihydropyridine KATP channel opener, [125I]A-312110 [(9R)-9-(4-fluoro-3-125iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano[3,4-b]thieno[2,3-e]pyridin-8(7H)-one-1,1-dioxide]. Binding of [125I]A-312110 to guinea pig cardiac (KD = 5.8 nM) and urinary bladder (KD = 4.9 nM) membranes were of high affinity, saturable, and to a single set of binding sites. Displacement of [125I]A-312110 by structurally diverse potassium channel openers (KCOs) indicated a similar rank order of potency in both guinea pig cardiac and bladder membranes (Ki, heart): A-312110 (4.3 nM)N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine (P1075)(-)-N-(2-ethoxyphenyl)-N'-(1,2,3-trimethylpropyl)-2-nitroethene-1,1-diamine (Bay X 9228)pinacidil(-)-cromakalimN-(4-benzoyl phenyl)-3,3,3-trifluro-2-hydroxy-2-methylpropionamine (ZD6169)9-(3-cyanophenyl)-3,4,6,7,9,10-hexahydro-1,8-(2H,5H)-acridinedione (ZM244085)diazoxide (16.7 microM). Displacement by KATP channel blockers, the sulfonylurea glyburide, and the cyanoguanidine N-[1-(3-chlorophenyl)cyclobutyl]-N'-cyano-N"-3-pyridinyl-guanidine (PNU-99963) were biphasic in the heart but monophasic in bladder with about a 100- to 500-fold difference in Ki values between high- and low-affinity sites. Good correlations were observed between cardiac or bladder-binding affinities of KCOs with functional activation as assessed by their respective potencies to either suppress action potential duration (APD) in Purkinje fibers or to relax electrical field-stimulated bladder contractions. Collectively, these results demonstrate that [125I]A-312110 binds with high affinity and has an improved activity profile compared with other radiolabeled KCOs. [125I]A-312110 is a useful tool for investigation of the molecular and functional properties of the KATP channel complex and for the identification, in a high throughput manner, of both novel channel blockers and openers that interact with cardiac/smooth muscle-type KATP channels.

Background Otilonium bromide (OB) is used as a spasmolytic drug in the treatment of the functional bowel disorder irritable bowel syndrome. Although its acute effects on colonic relaxation are well-characterized, little is known about the effects of chronic administration of OB on enteric neurons, neuromuscular transmission, and interstitial cells of Cajal (ICC), key regulators of the gut function. Methods Adult Sprague–Dawley rats were treated with OB in drinking water at a dose of 2 mg/kg for 30 days. The colons of OB-treated and age-matched control rats were studied by confocal immunohistochemistry to detect immunoreactivity (IR) in myenteric plexus neurons for nitrergic and tachykininergic markers, and also by microelectrode electrophysiology. Key Results Using immunohistochemistry, chronic OB administration did not change total neuron number, assessed by anti-Hu IR, but resulted in a significant increase in NK1 receptor positive neurons, a decrease in neuronal nitric oxide synthase expressing neurons, and a reduction in volume of substance P in nerve fibers in the myenteric plexus. Chronic OB administration potentiated inhibitory and excitatory junction potentials evoked by repetitive electrical field stimulation. The various types of colonic ICC, detected by Kit IR, were not altered nor were slow waves or smooth muscle membrane potential. Conclusions & Inferences Chronic treatment with OB caused significant changes in the nitrergic and tachykinergic components of the myenteric plexus and in both inhibitory and excitatory neurotransmission in the rat colon.

Interstitial cells of Cajal (ICC) form networks that intercalate between the enteric nervous system and smooth muscle cells and play a fundamental role in the control of gastrointestinal motility by initiating rhythmic electrical activity. In this report, we used a method to examine the physiological and morphological properties of ICC in living, intact tissues. ACK2, an anti-Kit antibody, was conjugated to a fluorescent probe and used to identify individual ICC for intracellular electrical recordings, to record changes in intracellular calcium concentration using fluorescent dyes and for confocal microscopy. Cyclic changes in intracellular calcium concentration were recorded in ICC with a frequency similar to the electrical slow wave. In addition, injection of a fluorescent dye into single ICC enabled the three-dimensional reconstruction of single myenteric plexus ICC within the intact network. The data show that ICC in intact networks from the myenteric plexus region in living tissues in the guinea-pig antrum exhibit an electrical slow wave, and that intracellular calcium oscillates at a frequency similar to the slow wave.

We investigated whether muscarinic receptor (MR) activation increases human urinary bladder smooth muscle (UBSM) excitability by directly or indirectly inhibiting the large conductance Ca2+-activat...

INTRODUCTION AND OBJECTIVES: Hyaluronic acid (HA), a non-sulfated glycosaminoglycan and an essential component of the extracellular matrix (ECM), plays critical roles in phases of wound healing and tissue regeneration. This study was designed to characterize temporal and anatomical HA deposition and its major receptors (CD44, LYVE-1, RHAMM, and HARE) expression in a rat bladder regeneration model and to understand the effectiveness of HA in promoting angiogenesis. METHODS: Fifteen Sprague-Dawley rats were subjected to partial cystectomy with interposition of distal sections of porcine small intestinal submucosa (SIS). SIS-augmented bladders were harvested on post-operative days 2, 7, 14, 28, and 56 for histological studies. Effectiveness of HA in stimulating endothelial cell proliferation, tube formation, and angiogenic activity were determined in vitro cell and ex ovo chick chorioallantoic membrane (CAM) models. RESULTS: Bladder regeneration proceeded without complications; and all regenerated bladders had complete urothelial lining and smooth muscle bundle formation by day 56 post-augmentation. As determined by immunohistochemistry, strong positive HA staining was observed on days 28 and 56 post-operation in the ECM of the stroma. CD44 immunoreactivity was detected on days 28 and 56 in the cytoplasm of urothelial cells and LYVE-1 immunoreactivity was detected exclusively in lymphatic vessels at days 28 and 56. RHAMM mRNA increased within 2 days post-operation, whereas HARE mRNA level did not change during the course of bladder regeneration. Exogenous HA significantly stimulated growth and tube formation of cultured endothelial cells, and enhanced angiogenesis in CAM assay. CONCLUSIONS: During bladder regeneration HA is synthesized throughout the course of regeneration processes. HA deposition coincides with urothelial differentiation. Temporal regulation and spatial distribution of two major HARs (CD44 and LYVE-1) follow the same temporal pattern as HA deposition. Therapeutic modalities through local delivery of exogenous HA can be viable to significantly improve angiogenesis and enhance the outcome and completeness of bladder regeneration.

INTRODUCTION AND OBJECTIVES: Hyaluronic acid (HA), a non-sulfated glycosaminoglycan and an essential component of the extracellular matrix (ECM), plays critical roles in phases of wound healing and tissue regeneration. This study was designed to characterize temporal and anatomical HA deposition and its major receptors (CD44, LYVE-1, RHAMM, and HARE) expression in a rat bladder regeneration model and to understand the effectiveness of HA in promoting angiogenesis. METHODS: Fifteen Sprague-Dawley rats were subjected to partial cystectomy with interposition of distal sections of porcine small intestinal submucosa (SIS). SIS-augmented bladders were harvested on post-operative days 2, 7, 14, 28, and 56 for histological studies. Effectiveness of HA in stimulating endothelial cell proliferation, tube formation, and angiogenic activity were determined in vitro cell and ex ovo chick chorioallantoic membrane (CAM) models. RESULTS: Bladder regeneration proceeded without complications; and all regenerated bladders had complete urothelial lining and smooth muscle bundle formation by day 56 post-augmentation. As determined by immunohistochemistry, strong positive HA staining was observed on days 28 and 56 post-operation in the ECM of the stroma. CD44 immunoreactivity was detected on days 28 and 56 in the cytoplasm of urothelial cells and LYVE-1 immunoreactivity was detected exclusively in lymphatic vessels at days 28 and 56. RHAMM mRNA increased within 2 days post-operation, whereas HARE mRNA level did not change during the course of bladder regeneration. Exogenous HA significantly stimulated growth and tube formation of cultured endothelial cells, and enhanced angiogenesis in CAM assay. CONCLUSIONS: During bladder regeneration HA is synthesized throughout the course of regeneration processes. HA deposition coincides with urothelial differentiation. Temporal regulation and spatial distribution of two major HARs (CD44 and LYVE-1) follow the same temporal pattern as HA deposition. Therapeutic modalities through local delivery of exogenous HA can be viable to significantly improve angiogenesis and enhance the outcome and completeness of bladder regeneration.

Purpose: Cavernous smooth muscle cells have a key role in the control of penile erection and detumescence. In this study the types of smooth muscle cells and currents present in isolated rabbit corpus cavernosum myocytes were characterized.Materials and Methods: Immunohistochemical methods were used to identify cavernous smooth muscle cells. Currents were recorded from freshly dissociated myocytes using the whole cell and amphotericin perforated patch clamp techniques.Results: Cavernous myocytes were identified by α-smooth muscle actin and smooth muscle myosin immunoreactivity. Based on electrical properties at least 2 types of myocytes were present. Type I cells showed more depolarized membrane potentials, lower capacitance, higher input resistance and increased current densities at positive potentials than type II cells. In types I and II cells at voltages positive to 30 mV, maxi K+ channel (Ca2+ activated large conductance K+ channel or BK) blockade with iberiotoxin or charybdotoxin reduced outward cur...

Purpose: The pathophysiology of diabetes mellitus induced erectile dysfunction is poorly understood. In patients with diffuse venous leakage structural changes in the corpora cavernosa have correlated with failure of the veno-occlusive mechanism. Three-dimensional (D) micro computerized tomography (CT) has proved to be an important imaging technique for the intact kidney, heart, liver and bone. We examined control and diabetic rabbit penises by 3-D micro CT and quantified any structural changes.Materials and Methods: Male white New Zealand rabbits were treated with alloxan to induce diabetes or used as normal controls. Via aortic access at laparotomy the penile vascular tree was vasodilated with papaverine and perfused with radiopaque silicone rubber. X-ray micro CT was then performed at 21 μm. resolution and images were analyzed in 3-D using custom software.Results: Nine diabetic rabbits with blood glucose greater than 400 mg./dl. and 9 control animals were used for micro CT analysis. Significant decreas...