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

2013 ◽  
Vol 304 (2) ◽  
pp. R155-R163 ◽  
Author(s):  
Rupal P. Soder ◽  
Shankar P. Parajuli ◽  
Kiril L. Hristov ◽  
Eric S. Rovner ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Electrical Field Stimulation ◽  
Tissue Level ◽  
Isometric Tension ◽  
Resting Membrane Potential ◽  
Sk Channels ◽  
Bladder Smooth Muscle ◽  
Patch Clamp Technique ◽  
Sk Channel ◽  
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 Ca2+-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.

scholarly journals Voltage-gated K+ channels sensitive to stromatoxin-1 regulate myogenic and neurogenic contractions of rat urinary bladder smooth muscle

2010 ◽  
Vol 299 (1) ◽  
pp. R177-R184 ◽  
Author(s):  
Muyan Chen ◽  
Whitney F. Kellett ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Urinary Bladder ◽  
Electrical Field Stimulation ◽  
Resting Membrane Potential ◽  
Bladder Smooth Muscle ◽  
Membrane Excitability ◽  
Molecular Fingerprints ◽  
Rat Urinary Bladder ◽  
Voltage Gated ◽  
Urinary Bladder Smooth Muscle

Members of the voltage-gated K+ (KV) channel family are suggested to control the resting membrane potential and the repolarization phase of the action potential in urinary bladder smooth muscle (UBSM). Recent studies report that stromatoxin-1, a peptide isolated from tarantulas, selectively inhibits KV2.1, KV2.2, KV4.2, and KV2.1/9.3 channels. The objective of this study was to investigate whether KV channels sensitive to stromatoxin-1 participate in the regulation of rat UBSM contractility and to identify their molecular fingerprints. Stromatoxin-1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and tone in isolated UBSM strips. However, stromatoxin-1 (100 nM) had no effect on the UBSM contractions induced by depolarizing agents such as KCl (20 mM) or carbachol (1 μM). This indicates that, under conditions of sustained membrane depolarization, the KV channels sensitive to stromatoxin-1 have no further contribution to the membrane excitability and contractility. Stromatoxin-1 (100 nM) increased the amplitude of the electrical field stimulation-induced contractions, suggesting also a role for these channels in neurogenic contractions. RT-PCR experiments on freshly isolated UBSM cells showed mRNA expression of KV2.1, KV2.2, and KV9.3, but not KV4.2 channel subunits. Protein expression of KV2.1 and KV2.2 channels was detected using Western blot and was further confirmed by immunocytochemical detection in freshly isolated UBSM cells. These novel findings indicate that KV2.1 and KV2.2, but not KV4.2, channel subunits are expressed in rat UBSM and play a key role in opposing both myogenic and neurogenic UBSM contractions.

scholarly journals Small-conductance, Ca2+-activated K+ channel 2 is the key functional component of SK channels in mouse urinary bladder

2008 ◽  
Vol 294 (5) ◽  
pp. R1737-R1743 ◽  
Author(s):  
K. S. Thorneloe ◽  
A. M. Knorn ◽  
P. E. Doetsch ◽  
E. S. R. Lashinger ◽  
A. X. Liu ◽  
...  
Keyword(s):  
Urinary Bladder ◽  
Action Potentials ◽  
Electrical Field Stimulation ◽  
K Channel ◽  
Sk Channels ◽  
Bladder Smooth Muscle ◽  
Sk Channel ◽  
Urinary Bladder Smooth Muscle ◽  
Insight Into ◽  
Small Conductance

Small-conductance Ca2+-activated K+ (SK) channels play an important role in regulating the frequency and in shaping urinary bladder smooth muscle (UBSM) action potentials, thereby modulating contractility. Here we investigated a role for the SK2 member of the SK family (SK1-3) utilizing: 1) mice expressing β-galactosidase (β-gal) under the direction of the SK2 promoter (SK2 β-gal mice) to localize SK2 expression and 2) mice lacking SK2 gene expression (SK2−/− mice) to assess SK2 function. In SK2 β-gal mice, UBSM staining was observed, but staining was undetected in the urothelium. Consistent with this, urothelial SK2 mRNA was determined to be 4% of that in UBSM. Spontaneous phasic contractions in wild-type (SK2+/+) UBSM strips were potentiated (259% of control) by the selective SK channel blocker apamin (EC50 = 0.16 nM), whereas phasic contractions of SK2−/− strips were unaffected. Nerve-mediated contractions of SK2+/+ UBSM strips were also increased by apamin, an effect absent in SK2−/− strips. Apamin increased the sensitivity of SK2+/+ UBSM strips to electrical field stimulation, since pretreatment with apamin decreased the frequency required to reach a 50% maximal contraction (vehicle, 21 ± 4 Hz, n = 6; apamin, 12 ± 2 Hz, n = 7; P < 0.05). In contrast, the sensitivity of SK2−/− UBSM strips was unaffected by apamin. Here we provide novel insight into the molecular basis of SK channels in the urinary bladder, demonstrating that the SK2 gene is expressed in the bladder and that it is essential for the ability of SK channels to regulate UBSM contractility.

scholarly journals Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels

2000 ◽  
Vol 279 (1) ◽  
pp. R60-R68 ◽  
Author(s):  
Gerald M. Herrera ◽  
Thomas J. Heppner ◽  
Mark T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Urinary Bladder ◽  
Negative Feedback ◽  
Ryanodine Receptors ◽  
Bk Channels ◽  
Sk Channels ◽  
Bladder Smooth Muscle ◽  
Contraction Amplitude ◽  
Contraction Frequency ◽  
Urinary Bladder Smooth Muscle

This study examines the roles of voltage-dependent Ca2+ channels (VDCC), ryanodine receptors (RyRs), large-conductance Ca2+-activated K+ (BK) channels, and small-conductance Ca2+-activated K+ (SK) channels in the regulation of phasic contractions of guinea pig urinary bladder smooth muscle (UBSM). Nisoldipine (100 nM), a dihydropyridine inhibitor of VDCC, abolished spontaneous UBSM contractions. Ryanodine (10 μM) increased contraction frequency and thereby integrated force and, in the presence of the SK blocker apamin, had a greater effect on integrated force than ryanodine alone. Blocking BK (iberiotoxin, 100 nM) or SK (apamin, 100 nM) channels increased contraction amplitude and duration but decreased frequency. The contractile response to iberiotoxin was more pronounced than to apamin. The increases in contraction amplitude and duration to apamin were substantially augmented with ryanodine pretreatment. These results indicate that BK and SK channels have prominent roles as negative feedback elements to limit UBSM contraction amplitude and duration. RyRs also appear to play a significant role as a negative feedback regulator of contraction frequency and duration, and this role is influenced by the activity of SK channels.

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 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.

Ca(2+)-activated K+ channels regulate action potential repolarization in urinary bladder smooth muscle

1997 ◽  
Vol 273 (1) ◽  
pp. C110-C117 ◽  
Author(s):  
T. J. Heppner ◽  
A. D. Bonev ◽  
M. T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Urinary Bladder ◽  
Action Potential ◽  
Single Channel ◽  
Resting Membrane Potential ◽  
Channel Blockers ◽  
Bladder Smooth Muscle ◽  
Apparent Dissociation Constant ◽  
K Channels ◽  
Urinary Bladder Smooth Muscle

The goal of this study was to examine the role of large conductance Ca(2+)-activated K+ channels in the regulation of cell excitability in urinary bladder smooth muscle from the guinea pig. Ca(2+)-activated K+ channels were studied with single-channel recording techniques and found to be intracellular Ca2+ and voltage dependent and sensitive to external tetraethylammonium and blocked by nanomolar concentrations of iberiotoxin (apparent dissociation constant of 4 nM). Spontaneous action potentials recorded from intact tissue strips depended on external Ca2+ and were inhibited by Ca2+ channel blockers. Iberiotoxin (100 nM) significantly altered the configuration of the action potential by increasing the duration and peak amplitude of the action potential and decreasing the rate of decay. Iberiotoxin also increased the action potential frequency from 0.11 to 0.29 Hz. This study suggests that Ca(2+)-activated K+ channels play a significant role in the repolarization of the action potential and in the maintenance of the resting membrane potential of the urinary bladder smooth muscle.

scholarly journals Nerve-released acetylcholine contracts urinary bladder smooth muscle by inducing action potentials independently of IP3-mediated calcium release

2010 ◽  
Vol 299 (3) ◽  
pp. R878-R888 ◽  
Author(s):  
Bernhard Nausch ◽  
Thomas J. Heppner ◽  
Mark T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Urinary Bladder ◽  
Action Potentials ◽  
Cyclopiazonic Acid ◽  
Electrical Field Stimulation ◽  
Field Stimulation ◽  
Bladder Smooth Muscle ◽  
Electrical Field ◽  
Urinary Bladder Smooth Muscle

Nerve-released ACh is the main stimulus for contraction of urinary bladder smooth muscle (UBSM). Here, the mechanisms by which ACh contracts UBSM are explored by determining Ca2+ and electrical signals induced by nerve-released ACh. Photolysis of caged inositol 1,4,5-trisphosphate (IP3) evoked Ca2+ release from the sarcoplasmic reticulum. Electrical field stimulation (20 Hz) induced Ca2+ waves within the smooth muscle that were present only during stimulus application. Ca2+ waves were blocked by inhibition of muscarinic ACh receptors (mAChRs) with atropine and depletion of sarcoplasmic reticulum Ca2+ stores with cyclopiazonic acid (CPA), and therefore likely reflect activation of IP3 receptors (IP3Rs). Electrical field stimulation also increased excitability to induce action potentials (APs) that were accompanied by Ca2+ flashes, reflecting Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs) during the action potential. The evoked Ca2+ flashes and APs occurred as a burst with a lag time of ∼1.5 s after onset of stimulation. They were not inhibited by blocking IP3-mediated Ca2+ waves, but by blockers of mAChRs (atropine) and VDCCs (diltiazem). Nerve-evoked contractions of UBSM strips were greatly reduced by blocking VDCCs, but not by preventing IP3-mediated Ca2+ signaling with cyclopiazonic acid or inhibition of PLC with U73122. These results indicate that ACh released from nerve varicosities induces IP3-mediated Ca2+ waves during stimulation; but contrary to expectations, these signals do not appear to participate in contraction. In addition, our data provide compelling evidence that UBSM contractions evoked by nerve-released ACh depend on increased excitability and the resultant Ca2+ entry through VDCCs during APs.

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

2013 ◽  
Vol 305 (2) ◽  
pp. C207-C214 ◽  
Author(s):  
Shankar P. Parajuli ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Selective Inhibition ◽  
Cellular Level ◽  
Bk Channels ◽  
Bk Channel ◽  
Bladder Smooth Muscle ◽  
Inhibitory Effects ◽  
Patch Clamp Technique ◽  
Rat Urinary Bladder ◽  
Urinary Bladder Smooth Muscle

Large conductance voltage- and Ca2+-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 Ca2+ 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 Ca2+. 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.

Negative feedback regulation of nerve-mediated contractions by KCa channels in mouse urinary bladder smooth muscle

2005 ◽  
Vol 289 (2) ◽  
pp. R402-R409 ◽  
Author(s):  
Gerald M. Herrera ◽  
Bud Etherton ◽  
Bernhard Nausch ◽  
Mark T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Urinary Bladder ◽  
Negative Feedback ◽  
Nerve Stimulation ◽  
Bk Channels ◽  
Bladder Smooth Muscle ◽  
Parasympathetic Nerves ◽  
Sk Channel ◽  
Urinary Bladder Smooth Muscle ◽  
Evoked Contractions

When the urinary bladder is full, activation of parasympathetic nerves causes release of neurotransmitters that induce forceful contraction of the detrusor muscle, leading to urine voiding. The roles of ion channels that regulate contractility of urinary bladder smooth muscle (UBSM) in response to activation of parasympathetic nerves are not well known. The present study was designed to characterize the role of large (BK)- and small-conductance (SK) Ca2+-activated K+ (KCa) channels in regulating UBSM contractility in response to physiological levels of nerve stimulation in UBSM strips from mice. Nerve-evoked contractions were induced by electric field stimulation (0.5–50 Hz) in isolated strips of UBSM. BK and SK channel inhibition substantially increased the amplitude of nerve-evoked contractions up to 2.45 ± 0.12- and 2.99 ± 0.25-fold, respectively. When both SK and BK channels were inhibited, the combined response was additive. Inhibition of L-type voltage-dependent Ca2+ channels (VDCCs) in UBSM inhibited nerve-evoked contractions by 92.3 ± 2.0%. These results suggest that SK and BK channels are part of two distinct negative feedback pathways that limit UBSM contractility in response to nerve stimulation by modulating the activity of VDCCs. Dysfunctional regulation of UBSM contractility by alterations in BK/SK channel expression or function may underlie pathologies such as overactive bladder.

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