Identifying the Active Sites in MoSi2@MoO3 Heterojunctions for Enhanced Hydrogen Evolution.

Developing Two-dimensional (2D) Mo-based heterogeneous nanomaterials is of great significance for energy conversion, especially in alkaline hydrogen evolution reaction (HER), however, it remains a challenge to identify the active sites at the interface due to the structure complexity. Herein, the real active sites are systematically explored during the HER process in varied Mo-based 2D materials by theoretical computational and magnetron sputtering approaches first to filtrate the candidates, then successfully combined the MoSi2 and MoO3 together through Oxygen doping to construct heterojunctions. Benefiting from the synergistic effects between the MoSi2 and MoO3, the obtained MoSi2@MoO3 exhibits an unprecedented overpotential of 72 mV at a current density of 10 mA cm-2. Density functional theory calculations uncover the different Gibbs free energy of hydrogen adsorption (ΔGH*) values achieved at the interfaces with different sites as adsorption sites. The results can facilitate the optimization of heterojunction electrocatalyst design principles for the Mo-based 2D materials.

Brain Structural Correlates of Dispositional Insight and the Mediation Role of Neuroticism in Young Adults.

Studies on the neural correlates of episodic insight have made significant progress in the past decades. However, the neural mechanisms underlying dispositional insight are largely unknown. In the present study, we recruited forty-four young, healthy adults and performed several analyses to reveal the neural mechanisms of dispositional insight. Firstly, a voxel-based morphometry (VBM) technique was used to explore the structural brain mechanisms of dispositional insight. We found that dispositional insight was significantly and negatively correlated with the regional gray matter volume (rGMV) in the left thalamus (TLM.L), right temporoparietal junction (TPJ.R), and left dorsal medial prefrontal cortex (DMPFC.L). Secondly, we performed a seed-based resting-state functional connectivity (RSFC) analysis to complement the findings of VBM analysis further. The brain regions of TLM.L, DMPFC.L, and TPJ.R were selected as seed regions. We found that dispositional insight was associated with altered RSFC between the DMPFC.L and bilateral TPJ, between the TPJ.R and left dorsolateral prefrontal cortex, left ventrolateral prefrontal cortex, DMPFC.L, TPJ.L, right insula, and right cerebellum. Finally, a mediation analysis found that the personality of neuroticism partially mediated the relationship between the brain region of TLM.L and dispositional insight. These findings imply that dispositional insight has a specific functional and structural neural mechanism. The personality of neuroticism may play a pivotal role in the processes of dispositional insight.

Neural responses to facial attractiveness in the judgments of moral goodness and moral beauty.

The judgments of moral goodness and moral beauty objectively refer to the perception and evaluation of moral traits, which are generally influenced by facial attractiveness. For centuries, people have equated beauty with the possession of positive qualities, but it is not clear whether the association between beauty and positive qualities exerts a similarly implicit influence on people's responses to moral goodness and moral beauty, how it affects those responses, and what is the neural basis for such an effect. The present study is the first to examine the neural responses to facial attractiveness in the judgments of moral goodness and moral beauty. We found that beautiful faces in both moral judgments activated the left ventral occipitotemporal cortices sensitive to the geometric configuration of the faces, demonstrating that both moral goodness and moral beauty required the automatic visual analysis of geometrical configuration of attractive faces. In addition, compared to beautiful faces during moral goodness judgment, beautiful faces during moral beauty judgment induced unique activity in the ventral medial prefrontal cortex and midline cortical structures involved in the emotional-valenced information about attractive faces. The opposite comparison elicited specific activity in the left superior temporal cortex and premotor area, which play a critical role in the recognition of facial identity. Our results demonstrated that the neural responses to facial attractiveness in the process of higher order moral decision-makings exhibit both task-general and task-specific characteristics. Our findings contribute to the understanding of the essence of the relationship between morality and aesthetics.

Neural underpinnings of morality judgment and moral aesthetic judgment.

Morality judgment usually refers to the evaluation of moral behavior`s ability to affect others` interests and welfare, while moral aesthetic judgment often implies the appraisal of moral behavior's capability to provide aesthetic pleasure. Both are based on the behavioral understanding. To our knowledge, no study has directly compared the brain activity of these two types of judgments. The present study recorded and analyzed brain activity involved in the morality and moral aesthetic judgments to reveal whether these two types of judgments differ in their neural underpinnings. Results reveled that morality judgment activated the frontal, parietal and occipital cortex previously reported for motor representations of behavior. Evaluation of goodness and badness showed similar patterns of activation in these brain regions. In contrast, moral aesthetic judgment elicited specific activations in the frontal, parietal and temporal cortex proved to be involved in the behavioral intentions and emotions. Evaluation of beauty and ugliness showed similar patterns of activation in these brain regions. Our findings indicate that morality judgment and moral aesthetic judgment recruit different cortical networks that might decode others' behaviors at different levels. These results contribute to further understanding of the essence of the relationship between morality judgment and aesthetic judgment.

Same-object costs and benefits in the object-based attentional blink.

Object-based attention (OBA) studies using the double-rectangle paradigm have revealed same-object costs with vertical rectangles but same-object benefits with horizontal rectangles, showing asymmetry in the OBA effect. Attentional blink (AB) studies using rapid serial visual presentation (RSVP) paradigms have shown that when two targets (T1 and T2) are successively presented within 400 msec, T2 performance is significantly impaired (i.e., an AB effect). Some object-based AB studies have indicated that the stable representation of single objects before T1 reduced the same-object AB effect, and some have claimed that the inhibition of the global objects representation by T1 processing enhanced the same-object AB effect. However, these studies did not directly explore whether and how the object representation modulated the object-based AB effect. In the present study, we used a hybrid of the RSVP and double-rectangle paradigms to address this issue. The results revealed that when the object representation was inhibited by T1 processing, the AB effect spread across the object groupings showing an enhanced same-object AB effect; when the object representation was created before T1 processing, there was a reliably reduced same-object AB effect that was not mediated by OBA (i.e., no difference in T2 performance between the same and different objects). Additionally, a horizontal configuration benefit independent of OBA was obtained in the AB.

Neural correlates of moral goodness and moral beauty judgments.

The objects of moral goodness and moral beauty judgments both generally refer to the positive moral acts or virtues of humans, and goodness must precede moral beauty. The main difference is that moral beauty, but not moral goodness, triggers emotional elevation. However, little is known about the neural mechanisms involved in both judgments. In the current study, 28 healthy female participants were scanned when they rated the good and beautiful extent of positive moral acts in daily life depicted in scene drawings to investigate the neural systems supporting moral goodness and moral beauty, specifically to test whether neural activity associated with moral beauty is same or different than moral goodness. The conjunction analysis of the contrasts between moral goodness judgment and moral beauty judgment identified the involvement of the left inferior orbitofrontal cortex (OFC), suggesting that the two judgments recruited the activity of a common brain region. Importantly, compared with the moral goodness judgment, the moral beauty judgment induced greater activity in more advanced cortical regions implicated in elevated emotions, including the anterior cingulate cortex (ACC), medial prefrontal cortex (mPFC), superior frontal gyrus (SFG) and the left temporo-parietal junction (TPJ). These regions have been strongly correlated with the cognitive aspects of moral cognition, including theory of mind (ToM). In addition, moral beauty judgment also activated brain regions implicated in empathy including the midline structures and the anterior insula. Based on these results, the brain harbors neural systems for common and for domain-specific evaluations of moral goodness and moral beauty judgments. Our study thus provides novel and compelling neural evidence for the essence of moral beauty and advances the current knowledge of the neural mechanisms underlying the beauty-is-good stereotype.

Author Correction: Different influences of facial attractiveness on judgments of moral beauty and moral goodness.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Different influences of facial attractiveness on judgments of moral beauty and moral goodness.

Are beauty and goodness the same? The relationship between beauty and goodness has long been a controversial issue in the fields of philosophy, aesthetics, ethics and psychology. Although many empirical studies have explored moral judgment and aesthetic judgment separately, only a few studies have compared the two. Whether these two judgments are two different processes or the same process with two different labels remains unclear. To answer this question, the present study directly compared the influence of facial attractiveness on judgments of moral goodness and moral beauty and revealed distinct contributions of imaging perceptions to these two judgments. The results showed that in the moral beauty judgment task, participants gave higher scores to characters with attractive faces compared with characters with unattractive faces, and larger P200 and LPP were elicited in the unattractive-face condition compared with the attractive-face condition; while in the moral goodness judgment task, there was no significant difference between the two conditions of either behaviour or ERP data. These findings offer important insights into the understanding and comparison of the processes of moral judgment and aesthetic judgment.

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.

Constitutive PKA activity is essential for maintaining the excitability and contractility in guinea pig urinary bladder smooth muscle: role of the BK channel.

The elevation of protein kinase A (PKA) activity activates the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels in urinary bladder smooth muscle (UBSM) cells and consequently attenuates spontaneous phasic contractions of UBSM. However, the role of constitutive PKA activity in UBSM function has not been studied. Here, we tested the hypothesis that constitutive PKA activity is essential for controlling the excitability and contractility of UBSM. We used patch clamp electrophysiology, line-scanning confocal and ratiometric fluorescence microscopy on freshly isolated guinea pig UBSM cells, and isometric tension recordings on freshly isolated UBSM strips. Pharmacological inhibition of the constitutive PKA activity with H-89 or PKI 14-22 significantly reduced the frequency and amplitude of spontaneous transient BK channel currents (TBKCs) in UBSM cells. Confocal and ratiometric fluorescence microscopy studies revealed that inhibition of constitutive PKA activity with H-89 reduced the frequency and amplitude of the localized Ca(2+) sparks but increased global Ca(2+) levels and the magnitude of Ca(2+) oscillations in UBSM cells. H-89 abolished the spontaneous transient membrane hyperpolarizations and depolarized the membrane potential in UBSM cells. Inhibition of PKA with H-89 or KT-5720 also increased the amplitude and muscle force of UBSM spontaneous phasic contractions. This study reveals the novel concept that constitutive PKA activity is essential for controlling localized Ca(2+) signals generated by intracellular Ca(2+) stores and cytosolic Ca(2+) levels. Furthermore, constitutive PKA activity is critical for mediating the spontaneous TBKCs in UBSM cells, where it plays a key role in regulating spontaneous phasic contractions in UBSM.

BK channel-mediated relaxation of urinary bladder smooth muscle: a novel paradigm for phosphodiesterase type 4 regulation of bladder function.

Elevation of intracellular cAMP and activation of protein kinase A (PKA) lead to activation of large conductance voltage- and Ca(2+)-activated K(+) (BK) channels, thus attenuation of detrusor smooth muscle (DSM) contractility. In this study, we investigated the mechanism by which pharmacological inhibition of cAMP-specific phosphodiesterase 4 (PDE4) with rolipram or Ro-20-1724 (C(15)H(22)N(2)O(3)) suppresses guinea pig DSM excitability and contractility. We used high-speed line-scanning confocal microscopy, ratiometric fluorescence Ca(2+) imaging, and perforated whole-cell patch-clamp techniques on freshly isolated DSM cells, along with isometric tension recordings of DSM isolated strips. Rolipram caused an increase in the frequency of Ca(2+) sparks and the spontaneous transient BK currents (TBKCs), hyperpolarized the cell membrane potential (MP), and decreased the intracellular Ca(2+) levels. Blocking BK channels with paxilline reversed the hyperpolarizing effect of rolipram and depolarized the MP back to the control levels. In the presence of H-89 [N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride], a PKA inhibitor, rolipram did not cause MP hyperpolarization. Rolipram or Ro-20-1724 reduced DSM spontaneous and carbachol-induced phasic contraction amplitude, muscle force, duration, and frequency, and electrical field stimulation-induced contraction amplitude, muscle force, and tone. Paxilline recovered DSM contractility, which was suppressed by pretreatment with PDE4 inhibitors. Rolipram had reduced inhibitory effects on DSM contractility in DSM strips pretreated with paxilline. This study revealed a novel cellular mechanism whereby pharmacological inhibition of PDE4 leads to suppression of guinea pig DSM contractility by increasing the frequency of Ca(2+) sparks and the functionally coupled TBKCs, consequently hyperpolarizing DSM cell MP. Collectively, this decreases the global intracellular Ca(2+) levels and DSM contractility in a BK channel-dependent manner.

Neurogenic detrusor overactivity is associated with decreased expression and function of the large conductance voltage- and Ca(2+)-activated K(+) channels.

Patients suffering from a variety of neurological diseases such as spinal cord injury, Parkinson's disease, and multiple sclerosis often develop neurogenic detrusor overactivity (NDO), which currently lacks a universally effective therapy. Here, we tested the hypothesis that NDO is associated with changes in detrusor smooth muscle (DSM) large conductance Ca(2+)-activated K(+) (BK) channel expression and function. DSM tissue samples from 33 patients were obtained during open bladder surgeries. NDO patients were clinically characterized preoperatively with pressure-flow urodynamics demonstrating detrusor overactivity, in the setting of a clinically relevant neurological condition. Control patients did not have overactive bladder and did not have a clinically relevant neurological disease. We conducted quantitative polymerase chain reactions (qPCR), perforated patch-clamp electrophysiology on freshly-isolated DSM cells, and functional studies on DSM contractility. qPCR experiments revealed that DSM samples from NDO patients showed decreased BK channel mRNA expression in comparison to controls. Patch-clamp experiments demonstrated reduced whole cell and transient BK currents (TBKCs) in freshly-isolated DSM cells from NDO patients. Functional studies on DSM contractility showed that spontaneous phasic contractions had a decreased sensitivity to iberiotoxin, a selective BK channel inhibitor, in DSM strips isolated from NDO patients. These results reveal the novel finding that NDO is associated with decreased DSM BK channel expression and function leading to increased DSM excitability and contractility. BK channel openers or BK channel gene transfer could be an alternative strategy to control NDO. Future clinical trials are needed to evaluate the value of BK channel opening drugs or gene therapies for NDO treatment and to identify any possible adverse effects.

Novel role of KT5720 on regulating hyperpolarization-activated cyclic nucleotide-gated channel activity and dorsal root ganglion neuron excitability.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in dorsal root ganglion (DRG) neurons, which are involved in diverse mechanisms that regulate DRG functions. Protein kinase A (PKA) is an essential kinase that plays a key role in almost all types of cells; it regulates the ion channel activity, the intracellular Ca(2+) concentration, as well as modulates cellular signals transduction. Nevertheless, the effect of PKA inhibition on the HCN channel activity in DRG neuron remains to be elucidated. Here we investigated the impact of PKA inhibition on the HCN channel activity and DRG neurons excitability. Our patch-clamp experiments both under whole-cell and single-channel conditions demonstrated that PKA inhibition with KT5720, a cell membrane permeable PKA-specific inhibitor, significantly attenuated HCN channel currents. Current clamp recording on freshly isolated DRG neurons showed KT5720 reduced overshoot amplitude and enhanced the threshold of the action potential. Moreover, our live-cell Ca(2+) imaging experiments illustrated KT5720 markedly reduced the intracellular Ca(2+) level. Collectively, this is the first report that addresses KT5720 attenuates the HCN channel activity and intracellular Ca(2+), thus reducing DRG neurons excitability. Therefore, our data strongly suggest that PKA is a potential target for curing HCN and DRG neuron relevant diseases.

TRPM4 channel: a new player in urinary bladder smooth muscle function in rats.

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.

Novel role for the transient potential receptor melastatin 4 channel in guinea pig detrusor smooth muscle physiology.

Members of the transient receptor potential (TRP) channel superfamily, including the Ca(2+)-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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.

Selective inhibition of phosphodiesterase 1 relaxes urinary bladder smooth muscle: role for ryanodine receptor-mediated BK channel activation.

The large conductance voltage- and Ca(2+)-activated K(+) (BK) channel is a major regulator of detrusor smooth muscle (DSM) excitability and contractility. Recently, we showed that nonselective phosphodiesterase (PDE) inhibition reduces guinea pig DSM excitability and contractility by increasing BK channel activity. Here, we investigated how DSM excitability and contractility changes upon selective inhibition of PDE type 1 (PDE1) and the underlying cellular mechanism involving ryanodine receptors (RyRs) and BK channels. PDE1 inhibition with 8-methoxymethyl-3-isobutyl-1-methylxanthine (8MM-IBMX; 10 µM) increased the cAMP levels in guinea pig DSM cells. Patch-clamp experiments on freshly isolated DSM cells showed that 8MM-IBMX increased transient BK currents and the spontaneous transient hyperpolarization (STH) frequency by ∼2.5- and ∼1.8-fold, respectively. 8MM-IBMX hyperpolarized guinea pig and human DSM cell membrane potential and significantly decreased the intracellular Ca(2+) levels in guinea pig DSM cells. Blocking BK channels with 1 µM paxilline or inhibiting RyRs with 30 µM ryanodine abolished the STHs and the 8MM-IBMX inhibitory effects on the DSM cell membrane potential. Isometric DSM tension recordings showed that 8MM-IBMX significantly reduced the spontaneous phasic contraction amplitude, muscle force integral, duration, frequency, and tone of DSM isolated strips. The electrical field stimulation-induced DSM contraction amplitude, muscle force integral, and duration were also attenuated by 10 µM 8MM-IBMX. Blocking BK channels with paxilline abolished the 8MM-IBMX effects on DSM contractions. Our data provide evidence that PDE1 inhibition relaxes DSM by raising cellular cAMP levels and subsequently stimulates RyRs, which leads to BK channel activation, membrane potential hyperpolarization, and decrease in intracellular Ca(2+) levels.

Constitutively active phosphodiesterase activity regulates urinary bladder smooth muscle function: critical role of KCa1.1 channel.

Pharmacological blockade of cyclic nucleotide phosphodiesterase (PDE) can relax human urinary bladder smooth muscle (UBSM); however, the underlying cellular mechanism is unknown. In this study, we investigated the effects of PDE pharmacological blockade on human UBSM excitability, spontaneous and nerve-evoked contractility, and determined the underlying cellular mechanism mediating these effects. Patch-clamp electrophysiological experiments showed that 3-isobutyl-1-methylxanthine (10 µM), a nonselective PDE inhibitor, caused ∼3.6-fold increase in the transient K(Ca)1.1 channel current frequency and ∼2.5-fold increase in the spontaneous transient hyperpolarization frequency in UBSM-isolated cells. PDE blockade also caused ∼5.6-mV hyperpolarization of the UBSM cell membrane potential. Blocking the K(Ca)1.1 channels with paxilline abolished the spontaneous transient hyperpolarization and the hyperpolarization effect of PDE blockade on the UBSM cell membrane potential. Live cell Ca(2+)-imaging experiments showed that PDE blockade significantly decreased the global intracellular Ca(2+) levels. Attenuation of PDE activity significantly reduced spontaneous phasic contraction amplitude, muscle force integral, duration, frequency, and muscle tone of human UBSM isolated strips. Blockade of PDE also significantly reduced the contraction amplitude, muscle force integral, and duration of the nerve-evoked contractions induced by 20-Hz electrical field stimulation. Pharmacological inhibition of K(Ca)1.1 channels abolished the relaxation effects of PDE blockade on both spontaneous and nerve-evoked contractions in human UBSM-isolated strips. Our data provide strong evidence that in human UBSM PDE is constitutively active, thus maintaining spontaneous UBSM contractility. PDE blockade causes relaxation of human UBSM by increasing transient K(Ca)1.1 channel current activity, hyperpolarizing cell membrane potential, and decreasing the global intracellular Ca(2+).

Expression and function of K(V)2-containing channels in human urinary bladder smooth muscle.

The functional role of the voltage-gated K(+) (K(V)) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of K(V)2.1, K(V)2.2, and the electrically silent K(V)9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of K(V)2.1, K(V)2.2, and K(V)4.2 homotetrameric channels and of K(V)2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca(2+) imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3 (but not K(V)4.2) channel subunits in human isolated DSM cells. K(V)2.1 and K(V)2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced K(V) current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca(2+) level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5-30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive K(V)2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

Suppression of human detrusor smooth muscle excitability and contractility via pharmacological activation of large conductance Ca2+-activated K+ channels.

Overactive bladder syndrome is frequently associated with increased detrusor smooth muscle (DSM) contractility. We tested the hypothesis that pharmacological activation of the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel with NS-1619, a selective BK channel opener, reduces the excitability and contractility of human DSM. We used the amphotericin-perforated whole cell patch-clamp technique on freshly isolated human DSM cells, live-cell Ca(2+) imaging, and isometric DSM tension recordings of human DSM strips obtained from open bladder surgeries. NS-1619 (30 µM) significantly increased the amplitude of the voltage step-induced whole cell BK currents, and this effect was abolished by pretreatment with 200 nM iberiotoxin (IBTX), a selective BK channel inhibitor. In current-clamp mode, NS-1619 (30 µM) significantly hyperpolarized the resting membrane potential, and the hyperpolarization was reversed by IBTX (200 nM). NS-1619 (30 µM) significantly decreased the intracellular Ca(2+) level in isolated human DSM cells. BK channel activation with NS-1619 (30 µM) significantly inhibited the amplitude, muscle force, frequency, duration, and tone of the spontaneous phasic and pharmacologically induced DSM contractions from human DSM isolated strips. IBTX (200 nM) suppressed the inhibitory effects of NS-1619 on spontaneous contractions. The amplitude of electrical field stimulation (0.5-50 Hz)-induced contractions was significantly reduced by NS-1619 (30 µM). Our data suggest that pharmacological activation of BK channels could represent a novel treatment option to control bladder dysfunction in humans.

Inhibition of phosphodiesterases relaxes detrusor smooth muscle via activation of the large-conductance voltage- and Ca²âº-activated K⁺ channel.

Detrusor smooth muscle (DSM) exhibits increased spontaneous phasic contractions under pathophysiological conditions such as detrusor overactivity (DO). Our previous studies showed that activation of cAMP signaling pathways reduces DSM contractility by increasing the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel activity. Here, we tested the hypothesis whether inhibition of phosphodiesterases (PDEs) can reduce guinea pig DSM excitability and contractility by increasing BK channel activity. Utilizing isometric tension recordings of DSM isolated strips and the perforated patch-clamp technique on freshly isolated DSM cells, we examined the mechanism of DSM relaxation induced by PDE inhibition. Inhibition of PDEs by 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor, significantly reduced DSM spontaneous and carbachol-induced contraction amplitude, frequency, duration, muscle force integral, and tone in a concentration-dependent manner. IBMX significantly reduced electrical field stimulation-induced contractions of DSM strips. Blocking BK channels with paxilline diminished the inhibitory effects of IBMX on DSM contractility, indicating a role for BK channels in DSM relaxation mediated by PDE inhibition. IBMX increased the transient BK currents (TBKCs) frequency by ∼3-fold without affecting the TBKCs amplitude. IBMX increased the frequency of the spontaneous transient hyperpolarizations by ∼2-fold and hyperpolarized the DSM cell resting membrane potential by ∼6 mV. Blocking the BK channels with paxilline abolished the IBMX hyperpolarizing effects. Under conditions of blocked Ca(2+) sources for BK channel activation, IBMX did not affect the depolarization-induced steady-state whole cell BK currents. Our data reveal that PDE inhibition with IBMX relaxes guinea pig DSM via TBKCs activation and subsequent DSM cell membrane hyperpolarization.