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

2012 ◽  
Vol 302 (11) ◽  
pp. C1632-C1641 ◽  
Author(s):  
Kiril L. Hristov ◽  
Shankar P. Parajuli ◽  
Rupal P. Soder ◽  
Qiuping Cheng ◽  
Eric S. Rovner ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Electrical Field Stimulation ◽  
Bk Channels ◽  
Bk Channel ◽  
Resting Membrane Potential ◽  
Force Frequency ◽  
Detrusor Smooth Muscle ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Current Clamp Mode

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 Ca2+-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 Ca2+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 Ca2+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.

scholarly journals Large-conductance voltage- and Ca2+-activated K+ channels regulate human detrusor smooth muscle function

2011 ◽  
Vol 301 (4) ◽  
pp. C903-C912 ◽  
Author(s):  
Kiril L. Hristov ◽  
Muyan Chen ◽  
Whitney F. Kellett ◽  
Eric S. Rovner ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Specific Inhibitor ◽  
Bk Channels ◽  
Bk Channel ◽  
Bladder Function ◽  
Detrusor Smooth Muscle ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Human Detrusor

The large-conductance voltage- and Ca2+-activated K+ (BK) channel is expressed in many smooth muscle types, but its role in human detrusor smooth muscle (DSM) is unclear. With a multidisciplinary approach spanning channel molecules, single-channel activity, freshly isolated human DSM cells, intact DSM preparations, and the BK channel specific inhibitor iberiotoxin, we elucidated human DSM BK channel function and regulation. Native human DSM tissues were obtained during open surgeries from patients with no preoperative history of overactive bladder. RT-PCR experiments on single human DSM cells showed mRNA expression of BK channel α-, β1-, and β4-subunits. Western blot and immunocytochemistry confirmed BK channel α, β1, and β4 protein expression. Native human BK channel properties were described using the perforated whole cell configuration of the patch-clamp technique. In freshly isolated human DSM cells, BK channel blockade with iberiotoxin inhibited a significant portion of the total voltage step-induced whole cell K+ current. From single BK channel recordings, human BK channel conductance was calculated to be 136 pS. Voltage-dependent iberiotoxin- and ryanodine-sensitive transient BK currents were identified in human DSM cells. In current-clamp mode, iberiotoxin inhibited the hyperpolarizing membrane potential transients and depolarized the cell resting membrane potential. Isometric DSM tension recordings revealed that BK channels principally control the contractions of isolated human DSM strips. Collectively, our results indicate that BK channels are fundamental regulators of DSM excitability and contractility and may represent new targets for pharmacological or genetic control of urinary bladder function in humans.

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

2016 ◽  
Vol 311 (6) ◽  
pp. F1253-F1259 ◽  
Author(s):  
Kiril L. Hristov ◽  
Shankar P. Parajuli ◽  
Aaron Provence ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Bladder Pressure ◽  
Resting Membrane Potential ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Whole Cell ◽  
Inside Out

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.

scholarly journals Inhibition of phosphodiesterases relaxes detrusor smooth muscle via activation of the large-conductance voltage- and Ca2+-activated K+ channel

2012 ◽  
Vol 302 (9) ◽  
pp. C1361-C1370 ◽  
Author(s):  
Wenkuan Xin ◽  
Qiuping Cheng ◽  
Rupal P. Soder ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Bk Channels ◽  
Bk Channel ◽  
Dependent Manner ◽  
Detrusor Smooth Muscle ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Pde Inhibition

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 Ca2+-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 Ca2+ 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.

scholarly journals Smooth muscle and neural mechanisms contributing to the downregulation of neonatal rat spontaneous bladder contractions during postnatal development

2007 ◽  
Vol 292 (5) ◽  
pp. R2100-R2112 ◽  
Author(s):  
Yuen-Keng Ng ◽  
William C. de Groat ◽  
Hsi-Yang Wu
Keyword(s):  
Smooth Muscle ◽  
Postnatal Development ◽  
Electrical Field Stimulation ◽  
Bk Channels ◽  
Bk Channel ◽  
Neonatal Rat ◽  
Field Stimulation ◽  
Electrical Field ◽  
Frequency Pattern ◽  
Rat Urinary Bladder

Spontaneous bladder contractions (SBCs) in the neonatal rat urinary bladder change from a high-amplitude, low-frequency pattern to a low-amplitude, high-frequency pattern during the first 6 wk of life. Understanding the mechanism of this developmental change may provide insights into the causes of bladder overactivity in adults. In vitro whole bladder preparations from Sprague-Dawley rats were used to study the modulation of SBCs by calcium-activated potassium channels (KCa) and electrical field stimulation from 3 days to 6 wk of life. SBCs in 3-day-old bladders were unmasked by treatment with iberiotoxin (100 nM), an inhibitor of large conductance KCa (BK) channels, or apamin (100 nM), an inhibitor of small conductance KCa (SK) channels. Iberiotoxin significantly increased the magnitude of SBCs at 2–3 wk, whereas apamin was only effective at 6 wk. In 1–2 wk bladders, exposure to room temperature Krebs solution decreased SBCs. This decrease was reversed by activating intramural nerves with electrical field stimulation. The effect of electrical field stimulation was inhibited by atropine (1 μM), suramin (10 μM), or pretreatment with tetrodotoxin (1 μM) but was not reversed by tetrodotoxin applied after electrical field stimulation. BK-α mRNA increased threefold, and BK-α protein increased fivefold from 3 days to 6 wk. These data suggest that BK channels play an important role in the regulation of SBCs in the neonatal bladder and that both increased BK channel activity, as well as changes in smooth muscle sensitivity to locally released neurotransmitters contribute to the downregulation of SBCs during early postnatal development.

scholarly journals Opposing roles of smooth muscle BK channels and ryanodine receptors in the regulation of nerve-evoked constriction of mesenteric resistance arteries

2014 ◽  
Vol 306 (7) ◽  
pp. H981-H988 ◽  
Author(s):  
Gayathri Krishnamoorthy ◽  
Swapnil K. Sonkusare ◽  
Thomas J. Heppner ◽  
Mark T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Cerebral Arteries ◽  
Ryanodine Receptors ◽  
Electrical Field Stimulation ◽  
Bk Channels ◽  
Bk Channel ◽  
Mesenteric Arteries ◽  
Resistance Arteries ◽  
Sympathetic Nerve Activation ◽  
Neurogenic Vasoconstriction

In depolarized smooth muscle cells of pressurized cerebral arteries, ryanodine receptors (RyRs) generate “Ca2+ sparks” that activate large-conductance, Ca2+-, and voltage-sensitive potassium (BK) channels to oppose pressure-induced (myogenic) constriction. Here, we show that BK channels and RyRs have opposing roles in the regulation of arterial tone in response to sympathetic nerve activation by electrical field stimulation. Inhibition of BK channels with paxilline increased both myogenic and nerve-induced constrictions of pressurized, resistance-sized mesenteric arteries from mice. Inhibition of RyRs with ryanodine increased myogenic constriction, but it decreased nerve-evoked constriction along with a reduction in the amplitude of nerve-evoked increases in global intracellular Ca2+. In the presence of L-type voltage-dependent Ca2+ channel (VDCC) antagonists, nerve stimulation failed to evoke a change in arterial diameter, and BK channel and RyR inhibitors were without effect, suggesting that nerve- induced constriction is dependent on activation of VDCCs. Collectively, these results indicate that BK channels and RyRs have different roles in the regulation of myogenic versus neurogenic tone: whereas BK channels and RyRs act in concert to oppose myogenic vasoconstriction, BK channels oppose neurogenic vasoconstriction and RyRs augment it. A scheme for neurogenic vasoregulation is proposed in which RyRs act in conjunction with VDCCs to regulate nerve-evoked constriction in mesenteric resistance arteries.

scholarly journals Spontaneous and evoked contractions are regulated by PKC-mediated signaling in detrusor smooth muscle: involvement of BK channels

2013 ◽  
Vol 304 (5) ◽  
pp. F451-F462 ◽  
Author(s):  
Joseph A. Hypolite ◽  
Qi Lei ◽  
Shaohua Chang ◽  
Stephen A. Zderic ◽  
Stephan Butler ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Outlet Obstruction ◽  
Bk Channels ◽  
Bk Channel ◽  
Quiescent State ◽  
Detrusor Smooth Muscle ◽  
Muscle Involvement ◽  
Normal Bladder ◽  
Spontaneous Contractions

Protein kinase C (PKC) and large conductance Ca2+-activated potassium channels (BK) are downregulated in the detrusor smooth muscle (DSM) in partial bladder outlet obstruction (PBOO). DSM from these bladders display increased spontaneous activity. This study examines the involvement of PKC in the regulation of spontaneous and evoked DSM contractions and whether pharmacologic inhibition of PKC in normal DSM contributes to increased detrusor excitability. Results indicate the PKC inhibitor bisindolylmaleimide 1 (Bim-1) prevented a decline in the amplitude of spontaneous DSM contractions over time in vitro, and these contractions persist in the presence of tetrodotoxin. Bim-1 also reduced the basal DSM tone, and the ability to maintain force in response to electrical field stimulation, but did not affect maximum contraction. The PKC activator phorbol-12,13-dibutyrate (PDBu) significantly reduced the amplitude and increased the frequency of spontaneous contractions at low concentrations (10 nM), while causing an increase in force at higher concentrations (1 μM). Preincubation of DSM strips with iberiotoxin prevented the inhibition of spontaneous contractions by PDBu. The BK channel openers isopimaric acid and NS1619 reduced the Bim-1-induced enhancement of spontaneous contractions in DSM strips. Our data suggest that PKC has a biphasic activation profile in the DSM and that it may play an important role in maintaining the quiescent state of the normal bladder during storage through the effects on BK channel, while helping to maintain force required for bladder emptying. The data also suggest that PKC dysfunction, as seen in PBOO, contributes to detrusor overactivity.

scholarly journals Ethanol-mediated relaxation of guinea pig urinary bladder smooth muscle: involvement of BK and L-type Ca2+ channels

2014 ◽  
Vol 306 (1) ◽  
pp. C45-C58 ◽  
Author(s):  
John Malysz ◽  
Serge A. Y. Afeli ◽  
Aaron Provence ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Urinary Bladder ◽  
Guinea Pig ◽  
Open Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Muscle Involvement ◽  
Whole Cell ◽  
Intracellular Ca ◽  
Probability Number

Mechanisms underlying ethanol (EtOH)-induced detrusor smooth muscle (DSM) relaxation and increased urinary bladder capacity remain unknown. We investigated whether the large conductance Ca2+-activated K+ (BK) channels or L-type voltage-dependent Ca2+ channels (VDCCs), major regulators of DSM excitability and contractility, are targets for EtOH by patch-clamp electrophysiology (conventional and perforated whole cell and excised patch single channel) and isometric tension recordings using guinea pig DSM cells and isolated tissue strips, respectively. EtOH at 0.3% vol/vol (∼50 mM) enhanced whole cell BK currents at +30 mV and above, determined by the selective BK channel blocker paxilline. In excised patches recorded at +40 mV and ∼300 nM intracellular Ca2+ concentration ([Ca2+]), EtOH (0.1–0.3%) affected single BK channels (mean conductance ∼210 pS and blocked by paxilline) by increasing the open channel probability, number of open channel events, and open dwell-time constants. The amplitude of single BK channel currents and unitary conductance were not altered by EtOH. Conversely, at ∼10 μM but not ∼2 μM intracellular [Ca2+], EtOH (0.3%) decreased the single BK channel activity. EtOH (0.3%) affected transient BK currents (TBKCs) by either increasing frequency or decreasing amplitude, depending on the basal level of TBKC frequency. In isolated DSM strips, EtOH (0.1–1%) reduced the amplitude and muscle force of spontaneous phasic contractions. The EtOH-induced DSM relaxation, except at 1%, was attenuated by paxilline. EtOH (1%) inhibited L-type VDCC currents in DSM cells. In summary, we reveal the involvement of BK channels and L-type VDCCs in mediating EtOH-induced urinary bladder relaxation accommodating alcohol-induced diuresis.

scholarly journals Expression and function of KV2-containing channels in human urinary bladder smooth muscle

2012 ◽  
Vol 302 (11) ◽  
pp. C1599-C1608 ◽  
Author(s):  
Kiril L. Hristov ◽  
Muyan Chen ◽  
Serge A. Y. Afeli ◽  
Qiuping Cheng ◽  
Eric S. Rovner ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Electrical Field Stimulation ◽  
Bladder Smooth Muscle ◽  
Rt Pcr ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

The functional role of the voltage-gated K+ (KV) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of KV2.1, KV2.2, and the electrically silent KV9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of KV2.1, KV2.2, and KV4.2 homotetrameric channels and of KV2.1/9.3 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 Ca2+ 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 KV2.1, KV2.2, and KV9.3 (but not KV4.2) channel subunits in human isolated DSM cells. KV2.1 and KV2.2 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 KV current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca2+ 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 KV2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

scholarly journals Chloroform Extract ofArtemisia annuaL. Relaxes Mouse Airway Smooth Muscle

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Jun Huang ◽  
Li-Qun Ma ◽  
Yongle Yang ◽  
Nana Wen ◽  
Wan Zhou ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Single Channel ◽  
Artemisia Annua ◽  
Chloroform Extract ◽  
Bk Channels ◽  
Bk Channel ◽  
Airway Smooth Muscle Cells ◽  
Patch Clamp Technique ◽  
Channel Currents

Artemisia annuaL. belongs to the Asteraceae family, which is indigenous to China. It has valuable pharmacological properties, such as antimalarial, anti-inflammatory, and anticancer properties. However, whether it possesses antiasthma properties is unknown. In the current study, chloroform extract ofArtemisia annuaL. (CEAA) was prepared, and we found that CEAA completely eliminated acetylcholine (ACh) or high K+-elicited (80 mM) contractions of mouse tracheal rings (TRs). Patch-clamp technique and ion channel blockers were employed to explore the underlying mechanisms of the relaxant effect of CEAA. In whole-cell current recording, CEAA almost fully abolished voltage-dependent Ca2+channel (VDCC) currents and markedly enhanced large conductance Ca2+-activated K+(BK) channel currents on airway smooth muscle cells (ASMCs). In single channel current recording, CEAA increased the opening probability but had no effect on the single channel conductance of BK channels. However, under paxilline-preincubated (a selective BK channel blocker) conditions, CEAA only slightly increased BK channel currents. These results indicate that CEAA may contain active components with potent antiasthma activity. The abolished VDCCs by CEAA may mainly contribute to the underlying mechanism through which it acts as an effective antiasthmatic compound, but the enhanced BK currents might play a less important role in the antiasthmatic effects.

scholarly journals Extracellular pH and intracellular phosphatidylinositol 4,5-bisphosphate control Cl− currents in guinea pig detrusor smooth muscle cells

2019 ◽  
Vol 317 (6) ◽  
pp. C1268-C1277 ◽  
Author(s):  
Viktor Yarotskyy ◽  
John Malysz ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Extracellular Ph ◽  
Complex Nature ◽  
Airway Smooth Muscle Cells ◽  
Detrusor Smooth Muscle ◽  
Patch Clamp Technique ◽  
Whole Cell

Cl− channels serve as key regulators of excitability and contractility in vascular, intestinal, and airway smooth muscle cells. We recently reported a Cl− conductance in detrusor smooth muscle (DSM) cells. Here, we used the whole cell patch-clamp technique to further characterize biophysical properties and physiological regulators of the Cl− current in freshly isolated guinea pig DSM cells. The Cl− current demonstrated outward rectification arising from voltage-dependent gating of Cl− channels rather than the Cl− transmembrane gradient. An exposure of DSM cells to hypotonic extracellular solution (Δ 165 mOsm challenge) did not increase the Cl− current providing strong evidence that volume-regulated anion channels do not contribute to the Cl− current in DSM cells. The Cl− current was monotonically dependent on extracellular pH, larger and lower in magnitude at acidic (5.0) and basic pH (8.5) values, respectively. Additionally, intracellularly applied phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] analog [PI(4,5)P2-diC8] increased the average Cl− current density by approximately threefold in a voltage-independent manner. The magnitude of the DSM whole cell Cl− current did not depend on the cell surface area (cell capacitance) regardless of the presence or absence of PI(4,5)P2-diC8, an intriguing finding that underscores the complex nature of Cl− channel expression and function in DSM cells. Removal of both extracellular Ca2+ and Mg2+ did not affect the DSM whole cell Cl− current, whereas Gd3+ (1 mM) potentiated the current. Collectively, our recent and present findings strongly suggest that Cl− channels are critical regulators of DSM excitability and are regulated by extracellular pH, Gd3+, and PI(4,5)P2.

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