Sequence divergence and retrotransposon insertion underlie interspecific epigenetic differences in primates.

Changes in the epigenome can affect the phenotype without the presence of changes in the genomic sequence. Given the high identity of the human and chimpanzee genome sequences, a substantial portion of their phenotypic divergence likely arises from epigenomic differences between the two species. In this study, the transcriptome and epigenome were determined for induced pluripotent stem cells (iPSCs) generated from human and chimpanzee individuals. The transcriptome and epigenomes for trimethylated histone H3 at lysine-4 (H3K4me3) and lysine-27 (H3K27me3) showed high levels of similarity between the two species. However, there were some differences in histone modifications. Although such regions, in general, did not show significant enrichment of interspecies nucleotide variations, gains in binding motifs for pluripotency-related transcription factors, especially POU5F1 and SOX2, were frequently found in species-specific H3K4me3 regions. We also revealed that species-specific insertions of retrotransposons, including the LTR5_Hs subfamily in human and a newly identified LTR5_Pt subfamily in chimpanzee, created species-specific H3K4me3 regions associated with increased expression of nearby genes. Human iPSCs have more species-specific H3K27me3 regions, resulting in more abundant bivalent domains. Only a limited number of these species-specific H3K4me3 and H3K27me3 regions overlap with species-biased enhancers in cranial neural crest cells, suggesting that differences in the epigenetic state of developmental enhancers appear late in development. Therefore, iPSCs serve as a suitable starting material for studying evolutionary changes in epigenome dynamics during development.

Enhancement of target toxin neutralization effect in vivo by PEGylation of multifunctionalized lipid nanoparticles.

Protein-protein (e.g., antibody-antigen) interactions comprise multiple weak interactions. We have previously reported that lipid nanoparticles (LNPs) bind to and neutralize target toxic peptides after multifunctionalization of the LNP surface (MF-LNPs) with amino acid derivatives that induce weak interactions; however, the MF-LNPs aggregated after target capture and showed short blood circulation times. Here we optimized polyethylene glycol (PEG)-modified MF-LNPs (PEG-MF-LNPs) to inhibit the aggregation and increase the blood circulation time. Melittin was used as a target toxin, and MF-LNPs were prepared with negatively charged, hydrophobic, and neutral amino-acid-derivative-conjugated functional lipids. In this study, MF-LNPs modified with only PEG5k (PEG5k-MF-LNPs) and with both PEG5k and PEG2k (PEGmix-MF-LNPs) were prepared, where PEG5k and PEG2k represent PEG with a molecular weight of 5000 and 2000, respectively. PEGylation of the MF-LNPs did not decrease the melittin neutralization ability of nonPEGylated MF-LNPs, as tested by hemolysis assay. The PEGmix-MF-LNPs showed better blood circulation characteristics than the PEG5k-MF-LNPs. Although the nonPEGylated MF-LNPs immediately aggregated when mixed with melittin, the PEGmix-MF-LNPs did not aggregate. The PEGmix-MF-LNPs dramatically increased the survival rate of melittin-treated mice, whereas the nonPEGylated MF-LNPs increased slightly. These results provide a fundamental strategy to improve the in vivo toxin neutralization ability of MF-LNPs.

Grinding and chemical mechanical polishing process for micropore x-ray optics fabricated with deep reactive ion etching.

Silicon micropore optics using deep reactive ion etching of silicon wafers has been being developed for future x-ray astronomy missions. Sidewalls of the micropores through a thin wafer with a typical thickness of hundreds of micrometers and a pore width of ∼20 µm are used for x-ray mirrors. However, burr structures observed after etching with a typical height of a few micrometers at the micropore edges are known to significantly reduce x-ray reflectivity. A new grinding and chemical mechanical polishing process is introduced to remove the burr structures. Both sides of the silicon wafer were ground and precisely polished after etching. X-ray reflectivity measurements confirmed an increase of reflectivity by 2-15 times at incident angles of 0.8-0.2 deg. The surface microroughness worsened from 2.0±0.2 nm rms to 7.8-0.8+0.6 nm rms; however, an additional annealing recovered the smooth surface and the estimated surface microroughness was <1.4 nm rms. This new process enables not only removing the burr structures but also choosing a flat part of the sidewalls for better angular resolution.

Tissue oxygen saturation levels from fetus to neonate.

AIM: Oxygen saturation during the term of delivery to the first cry, when fetal circulation dynamically changes, has not yet been examined. The aim of this study was therefore to determine whether the continuous measurement of regional tissue oxygen saturation (rSO2 ) from crowning until 5 min after delivery is possible using fetal tissue oximetry with a sensor attached to the examiner's finger. METHODS: Oxygen saturation levels in fetal cranial tissue between the second stage of delivery to crowning and up to 5 min after delivery were measured using fetal tissue oximetry with a sensor attached to the examiner's finger. Thirty-five deliveries were examined, and oxygen saturation was measured in seven infants from delivery of the head until 5 min after birth. Umbilical cord blood gas was measured in all cases. This clinical test was performed under the permission of the Ethics Committee of Hamamatsu University School of Medicine. RESULTS: Average tissue oxygen saturation in the second stage of delivery and at 5 min after delivery were 50.3 ± 16.3% and 56.8 ± 8.46%, respectively. In cases of continuous measurement, average rSO2 for crowning, immediately after delivery, and the first cry was 32.7 ± 9.5%, 30.0 ± 6.6%, and 31.6 ± 5.5%, respectively. CONCLUSION: We herein successfully measured oxygen saturation levels in fetal cranial tissue during crowning, delivery of the head, the first cry, and 5 min after delivery using fetal tissue oximetry with a sensor attached to the examiner's finger.

Examiner's finger-mounted fetal tissue oximetry: a preliminary report on 30 cases.

OBJECTIVE: To describe preliminary experience with a finger-mounted fetal tissue oximetry probe during the 2nd stage of labor. MATERIALS AND METHODS: A total of 30 term pregnant women without pregnancy complications were recruited. We measured fetal tissue oxygen saturation (FtO2) by using a finger-mounted fetal tissue oximetry during cervical examinations in the 2nd stage of labor. The data capturing rate of FtO2 and the interclass correlation coefficient were also examined. The mean FtO2 was compared to the neonatal condition assessed by the levels of umbilical cord blood. RESULTS: FtO2 was obtained in all cases, regardless of wetness, hair color, the part of the fetal head that was exposed, rotation of the fetus, color of amniotic fluid, and caput succedaneum. The mean FtO2 was 65.5%±8.58% in normal neonates [Apgar score >7 (1 min), n=25]. The mean FtO2 was significantly correlated with umbilical cord arterial pH (r=0.52, P=0.0030, n=30), but not with umbilical cord arterial partial pressure of oxygen. The interclass correlation coefficient was 0.94. CONCLUSIONS: Tissue oxygen saturation of the fetal head was obtained easily by the examiner's finger-mounted fetal tissue oximetry.

Myometrial relaxation of mice via expression of two pore domain acid sensitive K(+) (TASK-2) channels.

Myometrial relaxation of mouse via expression of two-pore domain acid sensitive (TASK) channels was studied. In our previous report, we suggested that two-pore domain acid-sensing K(+) channels (TASK-2) might be one of the candidates for the regulation of uterine circular smooth muscles in mice. In this study, we tried to show the mechanisms of relaxation via TASK-2 channels in marine myometrium. Isometric contraction measurements and patch clamp technique were used to verify TASK conductance in murine myometrium. Western blot and immunehistochemical study under confocal microscopy were used to investigate molecular identity of TASK channel. In this study, we showed that TEA and 4-AP insensitive non-inactivating outward K(+) current (NIOK) may be responsible for the quiescence of murine pregnant longitudinal myometrium. The characteristics of NIOK coincided with two-pore domain acid-sensing K(+) channels (TASK-2). NIOK in the presence of K(+) channel blockers was inhibited further by TASK inhibitors such as quinidine, bupivacaine, lidocaine, and extracellular acidosis. Furthermore, oxytocin and estrogen inhibited NIOK in pregnant myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed stronger inhibition of NIOK by quinidine and increased immunohistochemical expression of TASK-2. Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretch-activated channels in the longitudinal myometrium of mouse. Activation of TASK-2 channels seems to play an essential role for relaxing uterus during pregnancy and it might be one of the alternatives for preventing preterm delivery.

[A Japanese version of the FLANDERS handedness questionnaire].

Quantitative assessment of handedness is required in various clinical and research settings in psychology, neuroscience, and medicine. In the present study we tested the reliability and validity of a Japanese version of the FLANDERS handedness questionnaire, which was a new measure of skilled hand preference originally reported by Nicholls, Thomas, Loetscher, and Grimshaw (2013). Participants (N=431) completed three types of handedness questionnaires: the FLANDERS handedness questionnaire, Edinburgh Handedness Inventory, and H · N handedness test. Factor analysis revealed that the Japanese version of FLANDERS handedness questionnaire had a single-factor structure and high internal consistency. This questionnaire also posssed high test-retest reliability and criterion-referenced validity. These results indicate that the Japanese version of the FLANDERS handedness questionnaire is a valid and useful measure of skilled hand preference for Japanese participants.

Basic evaluation of the CRISPR/Cas system stability for application to paper-based analytical devices.

Despite the promising features of the CRISPR/Cas system for application to point-of-care nucleic acid tests, there are only a few reports on its integration into paper-based analytical devices (PADs) for the purpose of assay simplification. In most cases, paper platforms have only been used for the final signal readout in an assay otherwise performed in a test tube. Therefore, there is very limited information on the suitability of the CRISPR/Cas system for on-device reagent storage. To fill this gap, the current work primarily investigated the influence of various factors, including the type of paper, reagent drying method, effect of stabilizers, and storage condition on the storage stability of reagents necessary for CRISPR-based assays on paper substrates, by comparing the fluorescence signal emitted by the trans-cleavage of the dsDNA-activated Cas12a complex. The results obtained in the form of fluorescence signals emitted after trans-cleavage of a ssDNA probe through a dsDNA-activated Cas12a complex on paper substrates showed that CRISPR-related reagents spontaneously dried at room temperature on BSA blocked paper retained over 70% of their initial activity when stored at -20 °C for 28 days, independent of the type of paper substrates, which was improved by the addition of sucrose as a stabilizer. In addition, reagents dried on paper substrates under the optimized conditions exhibited stronger heat tolerance at temperatures above 65 °C compared to their corresponding solutions. This work is expected to contribute to the future development of fully integrated PADs relying on CRISPR/Cas systems for point-of-care applications requiring no additional reagent handling.

A case of leukemia cutis showing annular erythema during the course of Philadelphia chromosome-positive acute B-lymphoblastic leukemia.

We report a case of leukemia cutis showing annular erythema during the course of Philadelphia chromosome-positive acute B-lymphoblastic leukemia. The annular appearance may be developed by immunomodulatory effects of blinatumomab.

Mechanism of Relaxation Via TASK-2 Channels in Uterine Circular Muscle of Mouse.

Plasma pH can be altered during pregnancy and at labor. Membrane excitability of smooth muscle including uterine muscle is suppressed by the activation of K(+) channels. Because contractility of uterine muscle is regulated by extracellular pH and humoral factors, K(+) conductance could be connected to factors regulating uterine contractility during pregnancy. Here, we showed that TASK-2 inhibitors such as quinidine, lidocaine, and extracellular acidosis produced contraction in uterine circular muscle of mouse. Furthermore, contractility was significantly increased in pregnant uterine circular muscle than that of non-pregnant muscle. These patterns were not changed even in the presence of tetraetylammonium (TEA) and 4-aminopyridine (4-AP). Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretchactivated channels in myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed increased immunohistochemical expression of TASK-2. Therefore, TASK-2, seems to play a key role during regulation of myometrial contractility in the pregnancy and provides new insight into preventing preterm delivery.

LAG-3 expression in microglia regulated by IFN-γ/STAT1 pathway and metalloproteases.

Microglia are resident innate immune cells in the central nervous system (CNS) and play important roles in the development of CNS homeostasis. Excessive activation and neurotoxicity of microglia are observed in several CNS disorders, but the mechanisms regulating their activation remain unclear. Immune checkpoint molecules are expressed on activated immune cells and regulate their activation in peripheral immunity. However, the expression mechanism of immune checkpoint molecules in activated microglia is still unknown. Here, we analyzed the expression of immune checkpoint molecules in activated microglia using the mouse microglial cell line BV2 and primary cultured microglia. The expression of lymphocyte activation gene-3 (LAG-3), a type of immune checkpoint molecule, was increased in microglia activated by IFN-γ. IFN-γ-induced LAG-3 expression in microglia was suppressed by transfection of siRNA targeting STAT1. LAG-3 has two forms, membrane and soluble, and both forms were upregulated in microglia activated by IFN-γ. The production of soluble LAG-3 was suppressed by treatment with inhibitors of metalloproteinases such as ADAM10 and ADAM17. IFN-γ administration into cisterna magna of mice increased LAG-3 expression in spinal microglia. Furthermore, LAG-3 knockdown in microglia promoted nitric oxide production by IFN-γ. Our results demonstrate that LAG-3 expression in microglia is induced by the IFN-γ-STAT1 pathway and soluble LAG-3 production is regulated via cleavage of membranous LAG-3 by metalloproteinases including ADAM10 and ADAM17.

Predictive Modeling of Mental Illness Onset Using Wearable Devices and Medical Examination Data: Machine Learning Approach.

The prevention and treatment of mental illness is a serious social issue. Prediction and intervention, however, have been difficult because of lack of objective biomarkers for mental illness. The objective of this study was to use biometric data acquired from wearable devices as well as medical examination data to build a predictive model that can contribute to the prevention of the onset of mental illness. This was an observational study of 4,612 subjects from the health database of society-managed health insurance in Japan provided by JMDC Inc. The inputs to the predictive model were 3-months of continuous wearable data and medical examinations within and near that period; the output was the presence or absence of mental illness over the following month, as defined by insurance claims data. The features relating to the wearable data were sleep, activity, and resting heart rate, measured by a consumer-grade wearable device (specifically, Fitbit). The predictive model was built using the XGBoost algorithm and presented an area-under-the-receiver-operating-characteristic curve of 0.712 (SD = 0.02, a repeated stratified group 10-fold cross validation). The top-ranking feature importance measure was wearable data, and its importance was higher than the blood-test values from medical examinations. Detailed verification of the model showed that predictions were made based on disrupted sleep rhythms, mild physical activity duration, alcohol use, and medical examination data on disrupted eating habits as risk factors. In summary, the predictive model showed useful accuracy for grouping the risk of mental illness onset, suggesting the potential of predictive detection, and preventive intervention using wearable devices. Sleep abnormalities in particular were detected as wearable data 3 months prior to mental illness onset, and the possibility of early intervention targeting the stabilization of sleep as an effective measure for mental illness onset was shown.

Easy preparation of a liposome-mediated protein delivery system by freeze-thawing a liposome-protein complex.

Homeostasis can be achieved by adding a protein supplement; however, an appropriate vector is required to deliver the protein into the cell because of the low stability of proteins in the blood and low cell membrane permeability. Here we report an easy one-step method of encapsulating proteins into liposomes for delivery. We used negatively charged superoxide dismutase (SOD) and a polycation liposome as protein and liposome models, respectively. Liposome-encapsulated SOD was prepared by freeze-thawing the SOD-liposome complex (lipoplexes). The amount of immobilized SOD within the lipoplex significantly increased on freeze-thawing. Surprisingly, subjecting the single-layered lipoplexes to freeze-thawing produced multilayered liposomes with SOD localized between the lipid layers. The amount of SOD delivered intracellularly significantly increased by freeze-thawing compared with that delivered by lipoplexes without freeze-thawing. SOD, liposomes, and endosomes were separately localized in the cells. The freeze-thawed lipoplex-encapsulated SOD samples were intravenously injected in mice. The SOD biodistribution was dramatically changed compared with the injection of free SOD or lipoplex. SOD was detached from the lipoplex in the bloodstream after the injection of non-freeze-thawed lipoplex, whereas the encapsulation of SOD in the liposomes upon freeze-thawing enabled the stable circulation of SOD with the liposomes in the bloodstream. This work paves the way for the application of the freeze-thawing technology for the easy one-step encapsulation of proteins into liposomes for protein delivery.

Functional properties of suburothelial microvessels in the rat bladder.

PURPOSE: We investigated the properties of suburothelial microvessels, which have a vital role in maintaining microcirculation to cells involved in bladder afferent signaling. MATERIALS AND METHODS: Changes in the diameter of rat bladder suburothelial microvessels were measured using video microscopy. Membrane potential changes and intracellular Ca(2+) dynamics of suburothelial venules were examined using intracellular recording techniques and Ca(2+) imaging of fluo-4 fluorescence, respectively. RESULTS: Suburothelial venules showed spontaneous action potential and vasoconstriction activity while suburothelial arterioles were quiescent. Venular vasoconstriction was prevented by cyclopiazonic acid or nicardipine and decreased by 2-aminoethoxydiphenyl borate, niflumic acid or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Venular smooth muscle cells and perivascular interstitial cells showed spontaneous Ca(2+) transients. Nicardipine decreased the amplitude and disrupted the synchronicity of Ca(2+) transients in and between the 2 cell populations. Residual Ca(2+) transients in nicardipine occurred asynchronously and were abolished by cyclopiazonic acid. Suburothelial arterioles constricted in response to transmural nerve stimulation. These nerve induced constrictions were suppressed by prazosin or the selective α(1A) blocker RS17053 but not by the α(1D) blocker BMY7378. Remaining constrictions were abolished by guanethidine. CONCLUSIONS: Spontaneous vasoconstriction of suburothelial venules appears to result upon Ca(2+) release from the sarcoplasmic reticulum upon activation of inositol 1,4,5-trisphosphate receptors. This Ca(2+) opens Ca(2+) activated Cl(-) channels to trigger action potentials and Ca(2+) influx through L-type Ca(2+) channels. Adjacent perivascular interstitial cells may also have a role in generating this spontaneous venular vasoconstriction. In contrast, sympathetic nerve released noradrenaline acts on α(1A)-adrenoceptors to constrict suburothelial arterioles.

Properties of Rikkunshi-to (TJ-43)-induced relaxation of rat gastric fundus smooth muscles.

The relaxant effects of Rikkunshi-to (TJ-43), a gastroprotective herbal medicine, on rat gastric fundus were investigated. Experiments were carried out using standard tension and intracellular microelectrode recording techniques. During contraction induced by enprostil (0.5 microM), a prostaglandin E(2) analog, TJ-43, produced relaxation dose dependently (0.1-5.0 mg/ml) in the rat fundic circular smooth muscle (CSM) strips. The relaxant effects of TJ-43 were not affected by tetrodotoxin or 1 H[1, 2, 4] oxadiazolo [4, 3-a] quinoxalin-1-one (10 microM), an inhibitor of soluble guanylate cyclase. TJ-43 inhibited enprostil-induced membrane depolarization. Apamin (1 microM), a blocker of small-conductance Ca(2+)-activated K(+) (SK) channel, inhibited T-43-induced membrane repolarization. TJ-43-induced relaxation was biphasic, comprising of an initial fast followed by a second slow relaxation. The fast relaxation was abolished by apamin. Application of high K(+) (29.4 mM [K(+)](o)) also abolished the fast relaxation induced by TJ-43. In diabetic Goto-Kakizaki (GK) rat fundic CSM strips, the relaxant responses of TJ-43 during enprostil-induced contraction were increased compared with control rat strips. These results indicate that TJ-43 elicited fast muscle relaxation through membrane hyperpolarization induced by the activation of SK channels; the time-dependent slow relaxation reflects an additional direct of TJ-43 on CSM in the rat gastric fundus. Because TJ-43-evoked relaxation of fundic CSM strips was more potent in diabetic GK rat than in control rat, further analysis of this herb could lead to better treatments of diabetic gastroparesis.

Spontaneous electrical and Ca2+ signals in the mouse renal pelvis that drive pyeloureteric peristalsis.

1. Peristalsis in the smooth muscle cell (SMC) wall of the pyeloureteric system is unique in physiology in that the primary pacemaker resides in a population of atypical SMCs situated near the border of the renal papilla. 2. Atypical SMCs display high-frequency Ca(2+) transients upon the spontaneous release of Ca(2+) from inositol 1,4,5-trisphosphate (IP(3))-dependent stores that trigger cation-selective spontaneous transient depolarizations (STDs). In the presence of nifedipine, these Ca(2+) transients and STDs seldom propagate > 100 mum. Synchronization of STDs in neighbouring atypical SMCs into an electrical signal that can trigger action potential discharge and contraction in the typical SMC layer involves a coupled oscillator mechanism dependent on Ca(2+) entry through L-type voltage-operated Ca(2+) channels. 3. A population of spindle- or stellate-shaped cells, immunopositive for the tyrosine receptor kinase kit, is sparsely distributed throughout the pyeloureteric system. In addition, Ca(2+) transients and action potentials of long duration occurring at low frequencies have been recorded in a population of fusiform cells, which we have termed interstitial cells of Cajal (ICC)-like cells. 4. The electrical and Ca(2+) signals in ICC-like cells are abolished upon blockade of Ca(2+) release from either IP(3)- or ryanodine-dependent Ca(2+) stores. However, the spontaneous Ca(2+) signals in atypical SMCs or ICC-like cells are little affected in W/W(-v) transgenic mice, which have extensive lesions of their intestinal ICC networks. 5. In summary, we have developed a model of pyeloureteric pacemaking in which atypical SMCs are indeed the primary pacemakers, but the function of ICC-like cells has yet to be determined.

Correction: Filtered beauty in Oslo and Tokyo: A spatial frequency analysis of facial attractiveness.

[This corrects the article DOI: 10.1371/journal.pone.0227513.].

Filtered beauty in Oslo and Tokyo: A spatial frequency analysis of facial attractiveness.

Images of European female and male faces were digitally processed to generate spatial frequency (SF) filtered images containing only a narrow band of visual information within the Fourier spectrum. The original unfiltered images and four SF filtered images (low, medium-low, medium-high and high) were then paired in trials that kept constant SF band and face gender and participants made a forced-choice decision about the more attractive among the two faces. In this way, we aimed at identifying those specific SF bands where forced-choice preferences corresponded best to forced-choice judgements made when viewing the natural, broadband, facial images. We found that aesthetic preferences dissociated across SFs and face gender, but similarly for participants from Asia (Japan) and Europe (Norway). Specifically, preferences when viewing SF filtered images were best related to the preference with the broadband face images when viewing the highest filtering band for the female faces (about 48-77 cycles per face). In contrast, for the male faces, the medium-low SF band (about 11-19 cpf) related best to choices made with the natural facial images. Eye tracking provided converging evidence for the above, gender-related, SF dissociations. We suggest greater aesthetic relevance of the mobile and communicative parts for the female face and, conversely, of the rigid, structural, parts for the male face for facial aesthetics.

Interstitial cells of Cajal generate spontaneous transient depolarizations in the rat gastric fundus.

Intracellular recordings were made from isolated circular muscle bundles of rat gastric fundus. The majority of cells generated an ongoing discharge of electrical activity that were 15 min) resulted in the spread of dye between CSMC, between ICC-IM, and between CSMC and ICC-IM. Two types of STDs were observed, regularly occurring continuous STDs and irregular noisy bursting STDs. The amplitude of STDs varied between the two types of STDs. Single units summed to develop STDs with a maximum amplitude of 30 mV. Sodium nitroprusside (3 microM) induced membrane hyperpolarization and abolished unitary potentials generated by CSMC. In contrast, the amplitude of STDs generated by ICC-IM was increased with membrane hyperpolarization. Hyperpolarization induced by pinacidil (10 microM) also increased the amplitude of STDs and enhanced dV/dt(max). These observations indicate that STDs generated in ICC-IM spread passively to the adjacent CSMC to evoke the discharge of unitary potentials in the gastric fundus.

Role of K+ channels in regulating spontaneous activity in detrusor smooth muscle in situ in the mouse bladder.

PURPOSE: We investigated the functional role of K(+) channels for regulating spontaneous activity in mouse bladder detrusor smooth muscle. MATERIALS AND METHODS: The effects of different K(+) channels blockers on spontaneous changes in membrane potential and intracellular Ca(2+) dynamics were examined using intracellular recording techniques and Ca(2+) imaging with fluo-4 fluorescence, respectively. RESULTS: Detrusor smooth muscle generated spontaneous action potentials and Ca(2+) transients. Iberiotoxin (0.1 microM), charybdotoxin (0.1 microM) or tetraethylammonium (1 mM) increased the amplitude of action potentials and prolonged their repolarizing phase without inhibiting their after-hyperpolarization. Tetraethylammonium (10 mM) but not stromatoxin (0.1 microM) suppressed after-hyperpolarization and further increased the amplitude and half duration of action potentials. Apamin (0.1 microM) increased the frequency of action potentials but had no effect on their configuration. Spontaneous Ca(2+) transients were generated in individual detrusor smooth muscle cells and occasionally propagated to neighboring cells to form intercellular Ca(2+) waves. Transmural nerve stimulations invariably initiated synchronous Ca(2+) transients within and across muscle bundles. Charybdotoxin (0.1 microM) increased the amplitude of spontaneous Ca(2+) transients, while the subsequent application of tetraethylammonium (10 mM) increased their half duration. In addition, tetraethylammonium increased the synchronicity of Ca(2+) transients in muscle bundles. CONCLUSIONS: These results suggest that large and intermediate conductance Ca(2+) activated K(+) channels contribute to action potential repolarization and restrict the excitability of detrusor smooth muscle in the mouse bladder. In addition, the activation of voltage dependent K(+) channels is involved in repolarization and after-hyperpolarization, and it has a fundamental role in stabilizing detrusor smooth muscle excitability.