Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork.

Faithful DNA replication is essential for genome stability. To ensure accurate replication, numerous complex and redundant replication and repair mechanisms function in tandem with the core replication proteins to ensure DNA replication continues even when replication challenges are present that could impede progression of the replication fork. A unique topological challenge to the replication machinery is posed by RNA-DNA hybrids, commonly referred to as R-loops. Although R-loops play important roles in gene expression and recombination at immunoglobulin sites, their persistence is thought to interfere with DNA replication by slowing or impeding replication fork progression. Therefore, it is of interest to identify DNA-associated enzymes that help resolve replication-impeding R-loops. Here, using DNA fiber analysis, we demonstrate that human ribonuclease H1 (RNH1) plays an important role in replication fork movement in the mammalian nucleus by resolving R-loops. We found that RNH1 depletion results in accumulation of RNA-DNA hybrids, slowing of replication forks, and increased DNA damage. Our data uncovered a role for RNH1 in global DNA replication in the mammalian nucleus. Because accumulation of RNA-DNA hybrids is linked to various human cancers and neurodegenerative disorders, our study raises the possibility that replication fork progression might be impeded, adding to increased genomic instability and contributing to disease.

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

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

Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand.

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer.

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

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

Nascent peptides that block protein synthesis in bacteria.

Although the ribosome is a very general catalyst, it cannot synthesize all protein sequences equally well. For example, ribosomes stall on the secretion monitor (SecM) leader peptide to regulate expression of a downstream gene. Using a genetic selection in Escherichia coli, we identified additional nascent peptide motifs that stall ribosomes. Kinetic studies show that some nascent peptides dramatically inhibit rates of peptide release by release factors. We find that residues upstream of the minimal stalling motif can either enhance or suppress this effect. In other stalling motifs, peptidyl transfer to certain aminoacyl-tRNAs is inhibited. In particular, three consecutive Pro codons pose a challenge for elongating ribosomes. The translation factor elongation factor P, which alleviates pausing at polyproline sequences, has little or no effect on other stalling peptides. The motifs that we identified are underrepresented in bacterial proteomes and show evidence of stalling on endogenous E. coli proteins.

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.

Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle.

Large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca(2+) imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca(2+) sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca(2+) levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca(2+)-dependent mechanism, thus increasing DSM contractility.

Activation of muscarinic M3 receptors inhibits large-conductance voltage- and Ca2+-activated K+ channels in rat urinary bladder smooth muscle cells.

Large conductance voltage- and Ca(2+)-activated K(+) (BK) channels are key regulators of detrusor smooth muscle (DSM) contraction and relaxation during urine voiding and storage. Here, we explored whether BK channels are regulated by muscarinic receptors (M-Rs) in native freshly isolated rat DSM cells under physiological conditions using the perforated whole cell patch-clamp technique and pharmacological inhibitors. M-R activation with carbachol (1 µM) initially evoked large transient outward BK currents, followed by inhibition of the spontaneous transient outward BK currents (STBKCs) in DSM cells. Carbachol (1 µM) also inhibited the amplitude and frequency of spontaneous transient hyperpolarizations (STHs) and depolarized the DSM cell membrane potential. Selective inhibition of the muscarinic M3 receptors (M3-Rs) with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 0.1 µM), but not muscarinic M2 receptors with methoctramine (1 µM), blocked the carbachol inhibitory effects on STBKCs. Furthermore, blocking the inositol 1,4,5-triphosphate (IP3) receptors with xestospongin-C (1 µM) inhibited the carbachol-induced large transient outward BK currents without affecting carbachol inhibitory effects on STBKCs. Upon pharmacological inhibition of all known cellular sources of Ca(2+) for BK channel activation, carbachol (1 µM) did not affect the voltage-step-induced steady-state BK currents, suggesting that the muscarinic effects in DSM cells are mediated by mobilization of intracellular Ca(2+). In conclusion, our findings provide strong evidence that activation of M3-Rs leads to inhibition of the STBKCs, STHs, and depolarization of DSM cells. Collectively, the data suggest the existence of functional interactions between BK channels and M3-Rs at a cellular level in DSM.

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.

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.

SK channel-selective opening by SKA-31 induces hyperpolarization and decreases contractility in human urinary bladder smooth muscle.

Overactive bladder (OAB) is often associated with increased involuntary detrusor smooth muscle (DSM) contractions during the bladder-filling phase. To develop novel therapies for OAB, it is critical to better understand the mechanisms that control DSM excitability and contractility. Recent studies showed that small-conductance Ca(2+)-activated K(+) (SK) channels, SK3 channels, in particular, regulate human DSM function. However, the concept that SK channel-selective pharmacological activation can decrease the excitability and contractility directly in human DSM needs further exploration. Here, we studied the effect of the novel and potent SK channel activator, SKA-31 (or naphtho [1,2-d]thiazol-2-ylamine), on human DSM excitability and contractility at the cellular and tissue level. We used isometric tension recordings on human DSM-isolated strips and the perforated patch-clamp technique on freshly isolated native human DSM cells. SKA-31 significantly decreased spontaneous phasic contractions of DSM-isolated strips. In the presence of the SK channel blocker, apamin, the inhibitory effects of SKA-31 on the DSM spontaneous phasic contractions were significantly reduced. SKA-31 decreased the carbachol- and KCl-induced contractions in human DSM strips. Electrical field stimulation-induced contractions were significantly attenuated in the presence of SKA-31 at all stimulation frequencies (0.5-50 Hz). SKA-31 hyperpolarized the resting membrane potential of human DSM cells. Apamin abolished the hyperpolarizing effect of SKA-31, indicating the involvement of SK channel activation. These results support the concept that pharmacological activation of SK channels with selective openers may represent an attractive new pharmacological approach for decreasing DSM excitability and contractility, thus controlling OAB.

KV2.1 and electrically silent KV channel subunits control excitability and contractility of guinea pig detrusor smooth muscle.

Voltage-gated K(+) (K(V)) channels are implicated in detrusor smooth muscle (DSM) function. However, little is known about the functional role of the heterotetrameric K(V) channels in DSM. In this report, we provide molecular, electrophysiological, and functional evidence for the presence of K(V)2.1 and electrically silent K(V) channel subunits in guinea pig DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of the homotetrameric K(V)2.1, K(V)2.2, and K(V)4.2 as well as the heterotetrameric K(V)2.1/6.3 and K(V)2.1/9.3 channels, was used to examine the role of these K(V) channels in DSM function. RT-PCR indicated mRNA expression of K(V)2.1, K(V)6.2-6.3, K(V)8.2, and K(V)9.1-9.3 subunits in isolated DSM cells. K(V)2.1 protein expression was confirmed by Western blot and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the K(V) current in freshly isolated DSM cells. ScTx1 (100 nM) did not significantly change the steady-state activation and inactivation curves for K(V) current. However, ScTx1 (100 nM) decreased the activation time-constant of the K(V) current at positive voltages. Although our patch-clamp data could not exclude the presence of the homotetrameric K(V)2.1 channels, the biophysical characteristics of the ScTx1-sensitive current were consistent with the presence of heterotetrameric K(V)2.1/silent K(V) channels. Current-clamp recordings showed that ScTx1 (100 nM) did not change the DSM cell resting membrane potential. ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and muscle tone as well as the amplitude of the electrical field stimulation-induced contractions of isolated DSM strips. Collectively, our data revealed that K(V)2.1-containing channels are important physiological regulators of guinea pig DSM excitability and contractility.

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.

Pharmacological activation of small conductance calcium-activated potassium channels with naphtho[1,2-d]thiazol-2-ylamine decreases guinea pig detrusor smooth muscle excitability and contractility.

Small conductance Ca²âº-activated K⁺ (SK) and intermediate conductance Ca(2+)-activated K⁺ (IK) channels are thought to be involved in detrusor smooth muscle (DSM) excitability and contractility. Using naphtho[1,2-d]thiazol-2-ylamine (SKA-31), a novel and highly specific SK/IK channel activator, we investigated whether pharmacological activation of SK/IK channels reduced guinea pig DSM excitability and contractility. We detected the expression of all known isoforms of SK (SK1-SK3) and IK channels at mRNA and protein levels in DSM by single-cell reverse transcription-polymerase chain reaction and Western blot. Using the perforated patch-clamp technique on freshly isolated DSM cells, we observed that SKA-31 (10 µM) increased SK currents, which were blocked by apamin (1 µM), a selective SK channel inhibitor. In current-clamp mode, SKA-31 (10 µM) hyperpolarized the cell resting membrane potential, which was blocked by apamin (1 µM) but not by 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) (1 µM), a selective IK channel inhibitor. SKA-31 (10 nM-10 µM) significantly inhibited the spontaneous phasic contraction amplitude, frequency, duration, and muscle force in DSM isolated strips. The SKA-31 inhibitory effects on DSM contractility were blocked by apamin (1 µM) but not by TRAM-34 (1 µM), which did not per se significantly affect DSM spontaneous contractility. SK channel activation with SKA-31 reduced contractions evoked by electrical field stimulation. SKA-31 effects were reversible upon washout. In conclusion, SK channels, but not IK channels, mediate SKA-31 effects in guinea pig DSM. Pharmacological activation of SK channels reduces DSM excitability and contractility and therefore may provide a novel therapeutic approach for controlling bladder dysfunction.

Heterogeneous repolarization creates ventricular tachycardia circuits in healed myocardial infarction scar.

Arrhythmias originating in scarred ventricular myocardium are a major cause of death, but the underlying mechanism allowing these rhythms to exist remains unknown. This gap in knowledge critically limits identification of at-risk patients and treatment once arrhythmias become manifest. Here we show that potassium voltage-gated channel subfamily E regulatory subunits 3 and 4 (KCNE3, KCNE4) are uniquely upregulated at arrhythmia sites within scarred myocardium. Ventricular arrhythmias occur in areas with a distinctive cardiomyocyte repolarization pattern, where myocyte tracts with short repolarization times connect to myocytes tracts with long repolarization times. We found this unique pattern of repolarization heterogeneity only in ventricular arrhythmia circuits. In contrast, conduction abnormalities were ubiquitous within scar. These repolarization heterogeneities are consistent with known functional effects of KCNE3 and KCNE4 on the slow delayed-rectifier potassium current. We observed repolarization heterogeneity using conventional cardiac electrophysiologic techniques that could potentially translate to identification of at-risk patients. The neutralization of the repolarization heterogeneities could represent a potential strategy for the elimination of ventricular arrhythmia circuits.

Calcitonin gene-related peptide suppresses pacemaker currents by nitric oxide/cGMP-dependent activation of ATP-sensitive K(+) channels in cultured interstitial cells of Cajal from the mouse small intestine.

The effects of calcitonin gene-related peptide (CGRP) on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine were investigated using the whole-cell patch clamp technique at 30 degrees . Under voltage clamping at a holding potential of -70 mV, CGRP decreased the amplitude and frequency of pacemaker currents and activated outward resting currents. These effects were blocked by intracellular GDPbetaS, a G-protein inhibitor and glibenclamide, a specific ATP-sensitive K(+) channels blocker. During current clamping, CGRP hyperpolarized the membrane and this effect was antagonized by glibenclamide. Pretreatment with SQ-22536 (an adenylate cyclase inhibitor) or naproxen (a cyclooxygenase inhibitor) did not block the CGRP-induced effects, whereas pretreatment with ODQ (a guanylate cyclase inhibitor) or L-NAME (an inhibitor of nitric oxide synthase) did. In conclusion, CGRP inhibits pacemaker currents in ICC by generating nitric oxide via G-protein activation and so activating ATP-sensitive K(+) channels. Nitric oxide- and guanylate cyclase- dependent pathways are involved in these effects.

c-Myb and C/EBPß regulate OPN and other senescence-associated secretory phenotype factors.

Tumorigenesis results from the convergence of cell autonomous mutations and corresponding stromal changes that promote tumor cell growth. Senescent cells, which secrete a plethora of pro-tumorigenic factors termed the senescence-associated secretory phenotype (SASP), play an important role in tumor formation. Investigation into SASP regulation revealed that many but not all SASP factors are subject to NF-kB and p38MAPK regulation. However, many pro-tumorigenic SASP factors, including osteopontin (OPN), are not responsive to these canonical pathways leaving the regulation of these factors an open question. We report that the transcription factor c-Myb regulates OPN, IL-6, and IL-8 in addition to 57 other SASP factors. The regulation of OPN is direct as c-Myb binds to the OPN promoter in response to senescence. Further, OPN is also regulated by the known SASP regulator C/EBPß. In response to senescence, the full-length activating C/EBPß isoform LAP2 increases binding to the OPN, IL-6, and IL-8 promoters. The importance of both c-Myb and C/EBPß is underscored by our finding that the depletion of either factor reduces the ability of senescent fibroblasts to promote the growth of preneoplastic epithelial cells.

Effects of ATP on Pacemaker Activity of Interstitial Cells of Cajal from the Mouse Small Intestine.

Purinergic receptors play an important role in regulating gastrointestinal (GI) motility. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate GI smooth muscle activity. We studied the functional roles of external adenosine 5'-triphosphate (ATP) on pacemaker activity in cultured ICCs from mouse small intestines by using the whole-cell patch clamp technique and intracellular Ca2+ ([Ca2+]i) imaging. External ATP dose-dependently depolarized the resting membrane and produced tonic inward pacemaker currents, and these effects were antagonized by suramin, a purinergic P2 receptor antagonist. ATP-induced effects on pacemaker currents were suppressed by an external Na+-free solution and inhibited by the nonselective cation channel blockers, flufenamic acid and niflumic acid. The removal of external Ca2+ or treatment with thapsigargin (inhibitor of Ca2+ uptake into endoplasmic reticulum) inhibited the ATP-induced effects on pacemaker currents. Spontaneous [Ca2+]i oscillations were enhanced by external ATP. These results suggest that external ATP modulates pacemaker activity by activating nonselective cation channels via external Ca2+ influx and [Ca2+]i release from the endoplasmic reticulum. Thus, it seems that activating the purinergic P2 receptor may modulate GI motility by acting on ICCs in the small intestine.

Inhibition of pacemaker currents by nitric oxide via activation of ATP-sensitive K+ channels in cultured interstitial cells of Cajal from the mouse small intestine.

We investigated the role of nitric oxide (NO) in pacemaker activity and signal mechanisms in cultured interstitial cells of Cajal (ICC) of the mouse small intestine using whole cell patch-clamp techniques at 30 degrees C. ICC generated pacemaker potential in the current clamp mode and pacemaker currents at a holding potential of -70 mV. (+/-)-S-nitroso-N-acetylpenicillamine (SNAP; a NO donor) produced membrane hyperpolarization and inhibited the amplitude and frequency of the pacemaker currents, and increased resting currents in the outward direction. These effects were blocked by the use of glibenclamide (an ATP-sensitive K+ channel blocker), but not by the use of 5-hydroxydecanoic acid (a mitochondrial ATP-sensitive K+ channel blocker). Pretreatment with ODQ (a guanylate cyclase inhibitor) almost blocked the NO-induced effects. The use of cell-permeable 8-bromo-cyclic GMP also mimicked the action of SNAP. However, the use of KT-5823 (a protein kinase G inhibitor) did not block the NO-induced effects. Spontaneous [Ca2+]i oscillations in ICC were inhibited by the treatment of SNAP, as seen in recordings of intracellular Ca2+ ([Ca2+]i). These results suggest that NO inhibits pacemaker activity by the activation of ATP-sensitive K+ channels via a cyclic GMP dependent mechanism in ICC, and the activation of ATP-sensitive K+ channels mediates the inhibition of spontaneous [Ca2+]i oscillations.

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

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