Effects of the gap junction blocker glycyrrhetinic acid on gastrointestinal smooth muscle cells

2005 ◽  
Vol 288 (4) ◽  
pp. G832-G841 ◽  
Author(s):  
Yukari Takeda ◽  
Sean M. Ward ◽  
Kenton M. Sanders ◽  
Sang Don Koh
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Gap Junctions ◽  
Lucifer Yellow ◽  
Muscle Cells ◽  
Glycyrrhetinic Acid ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
Patch Clamp Technique

In the tunica muscularis of the gastrointestinal (GI) tract, gap junctions form low-resistance pathways between pacemaker cells known as interstitial cells of Cajal (ICCs) and between ICC and smooth muscle cells. Coupling via these junctions facilitates electrical slow-wave propagation and responses of smooth muscle to enteric motor nerves. Glycyrrhetinic acid (GA) has been shown to uncouple gap junctions, but previous studies have shown apparent nonspecific effects of GA in a variety of tissues. We tested the effects of GA using isometric force measurements, intracellular microelectrode recordings, the patch-clamp technique, and the spread of Lucifer yellow within cultured ICC networks. In murine small intestinal muscles, β-GA (10 μM) decreased phasic contractions and depolarized resting membrane potential. Preincubation of GA inhibited the spread of Lucifer yellow, increased input resistance, and decreased cell capacitance in ICC networks, suggesting that GA uncoupled ICCs. In patch-clamp experiments of isolated jejunal myocytes, GA significantly decreased L-type Ca2+ current in a dose-dependent manner without affecting the voltage dependence of this current. The IC50 for Ca2+ currents was 1.9 μM, which is lower than the concentrations used to block gap junctions. GA also significantly increased large-conductance Ca2+-activated K+ currents but decreased net delayed rectifier K+ currents, including 4-aminopyridine and tetraethylammonium-resistant currents. In conclusion, the reduction of phasic contractile activity of GI muscles by GA is likely a consequence of its inhibitory effects on gap junctions and voltage-dependent Ca2+ currents. Membrane depolarization may be a consequence of uncoupling effects of GA on gap junctions between ICCs and smooth muscles and inhibition of K+ conductances in smooth muscle cells.

scholarly journals Contribution of Kv2.1 channels to the delayed rectifier current in freshly dispersed smooth muscle cells from rabbit urethra

2011 ◽  
Vol 301 (5) ◽  
pp. C1186-C1200 ◽  
Author(s):  
B. Kyle ◽  
E. Bradley ◽  
S. Ohya ◽  
G. P. Sergeant ◽  
N. G. McHale ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Hek 293 ◽  
Rabbit Urethra ◽  
Urethral Smooth Muscle

We have characterized the native voltage-dependent K+ (Kv) current in rabbit urethral smooth muscle cells (RUSMC) and compared its pharmacological and biophysical properties with Kv2.1 and Kv2.2 channels cloned from the rabbit urethra and stably expressed in human embryonic kidney (HEK)-293 cells (HEKKv2.1 and HEKKv2.2). RUSMC were perfused with Hanks′ solution at 37°C and studied using the patch-clamp technique with K+-rich pipette solutions. Cells were bathed in 100 nM Penitrem A (Pen A) to block large-conductance Ca2+-activated K+ (BK) currents and depolarized to +40 mV for 500 ms to evoke Kv currents. These were unaffected by margatoxin, κ-dendrotoxin, or α-dendrotoxin (100 nM, n = 3–5) but were blocked by stromatoxin-1 (ScTx, IC50 ∼130 nM), consistent with the idea that the currents were carried through Kv2 channels. RNA was detected for Kv2.1, Kv2.2, and the silent subunit Kv9.3 in urethral smooth muscle. Immunocytochemistry showed membrane staining for both Kv2 subtypes and Kv9.3 in isolated RUSMC. HEKKv2.1 and HEKKv2.2 currents were blocked in a concentration-dependent manner by ScTx, with estimated IC50 values of ∼150 nM (Kv2.1, n = 5) and 70 nM (Kv2.2, n = 6). The mean half-maximal voltage ( V1/2) of inactivation of the USMC Kv current was −56 ± 3 mV ( n = 9). This was similar to the HEKKv2.1 current (−55 ± 3 mV, n = 13) but significantly different from the HEKKv2.2 currents (−30 ± 3 mV, n = 11). Action potentials (AP) evoked from RUSMC studied under current-clamp mode were unaffected by ScTx. However, when ScTx was applied in the presence of Pen A, the AP duration was significantly prolonged. Similarly, ScTx increased the amplitude of spontaneous contractions threefold, but only after Pen A application. These data suggest that Kv2.1 channels contribute significantly to the Kv current in RUSMC.

Ketamine blocks Ca2+-activated K+ channels in rabbit cerebral arterial smooth muscle cells

2003 ◽  
Vol 285 (3) ◽  
pp. H1347-H1355 ◽  
Author(s):  
Jin Han ◽  
Nari Kim ◽  
Hyun Joo ◽  
Euiyong Kim
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Cerebral Arteries ◽  
Muscle Cells ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Arterial Smooth Muscle ◽  
Clamp Technique ◽  
Arterial Smooth Muscle Cells

Although ketamine and Ca2+-activated K+ (KCa) channels have been implicated in the contractile activity regulation of cerebral arteries, no studies have addressed the specific interactions between ketamine and the KCa channels in cerebral arteries. The purpose of this study was to examine the direct effects of ketamine on KCa channel activities using the patch-clamp technique in single-cell preparations of rabbit middle cerebral arterial smooth muscle. We tested the hypothesis that ketamine modulates the KCa channel activity of the cerebral arterial smooth muscle cells of the rabbit. Vascular myocytes were isolated from rabbit middle cerebral arteries using enzymatic dissociation. Single KCa channel activities of smooth muscle cells from rabbit cerebral arteries were recorded using the patch-clamp technique. In the inside-out patches, ketamine in the micromolar range inhibited channel activity with a half-maximal inhibition of the ketamine conentration value of 83.8 ± 12.9 μM. The Hill coefficient was 1.2 ± 0.3. The slope conductance of the current-voltage relationship was 320.1 ± 2.0 pS between 0 and +60 mV in the presence of ketamine and symmetrical 145 mM K+. Ketamine had little effect on either the voltage-dependency or open- and closed-time histograms of KCa channel. The present study clearly demonstrates that ketamine inhibits KCa channel activities in rabbit middle cerebral arterial smooth muscle cells. This inhibition of KCa channels may represent a mechanism for ketamine-induced cerebral vasoconstriction.

Effects of prostaglandin F2α on membrane currents in rabbit middle cerebral arterial smooth muscle cells

2003 ◽  
Vol 284 (3) ◽  
pp. H1018-H1027 ◽  
Author(s):  
Nari Kim ◽  
Jin Han ◽  
Euiyong Kim
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Smooth Muscles ◽  
Muscle Cells ◽  
Membrane Potentials ◽  
Delayed Rectifier ◽  
Direct Effects ◽  
Patch Clamp Technique ◽  
Arterial Smooth Muscle ◽  
Arterial Smooth Muscle Cells

Although PGF2αaffects contractility of vascular smooth muscles, no studies to date have addressed the electrophysiological mechanism of this effect. The purpose of our investigation was to examine the direct effects of PGF2α on membrane potentials, Ca2+-activated K+ (KCa) channels, delayed rectifier K+ (KV) channels, and L-type Ca2+channels with the patch-clamp technique in single rabbit middle cerebral arterial smooth muscle cells (SMCs). PGF2αsignificantly hyperpolarized membrane potentials and increased the amplitudes of total K+ currents. PGF2αincreased open-state probability but had little effect on the open and closed kinetics of KCa channels. PGF2αincreased the amplitudes of KV currents with a leftward shift of the activation and inactivation curves and a decrease in the activation time constant. PGF2α decreased the amplitudes of L-type Ca2+ currents without any significant change in threshold or apparent reversal potentials. This study provides the first finding that the direct effects of PGF2α on middle cerebral arterial SMCs, at least in part, could attenuate vasoconstriction.

ATP-sensitive potassium channels in cultured arterial segments

1996 ◽  
Vol 271 (6) ◽  
pp. H2462-H2468 ◽  
Author(s):  
T. Kleppisch ◽  
B. Winter ◽  
M. T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Katp Channels ◽  
Muscle Cells ◽  
Mesenteric Arteries ◽  
Force Density ◽  
Dependent Manner ◽  
Growth Responses ◽  
Patch Clamp Technique ◽  
Arterial Smooth Muscle

Organ cultures of arteries have been used to study growth responses, proliferation, and contractility. However, the function of specific-ion channels in cultured arteries has not been investigated. ATP-sensitive K+ (KATP) channels play an important role in the control of arterial tone. The goal of this study was to determine the functional state of KATP channels in arteries kept in culture. Segments from rabbit mesenteric arteries were cultured in for 2-7 days. To explore the properties of KATP channels, the effects of KATP-channel modulators and other vasoactive substances on isometric force, density, and modulation of KATP currents in single smooth muscle cells isolated from cultured vessels were examined. Isometric contractions were measured with a resistance-vessel myograph. Whole cell KATP currents were recorded with the patch-clamp technique. Membrane capacitance and KATP-current density in single smooth muscle cells from freshly dissected (control) and cultured arteries were not altered. At -60 mV, glibenclamide-sensitive currents in the presence of the K(+)-channel opener pinacidil were -4.7 +/- 1.2, -4.7 +/- 0.6, and -4.6 +/- 0.7 pA/pF for control and 2- and 4-day arteries, respectively. Inhibitory modulation of KATP currents in arterial smooth muscle also remained intact for 4 days in culture; the vasoconstrictor histamine (10 microM) reduced glibenclamide-sensitive currents in the presence of pinacidil by 61.2 +/- 2.8, 42.4 +/- 10.1, and 41.2 +/- 6.1% for control and 2- and 4-day arteries, respectively. Pinacidil relaxed control and cultured arteries (1-7 days) in a dose-dependent manner. Half-maximal effective concentrations of pinacidil were 0.42, 0.24, 0.23, and 0.51 microM for control and 2-, 4-, and 7-day arteries, respectively, whereas maximal relaxations to pinacidil were 62.9, 47.5, 37.5, and 55.7% for control and 2-, 5-, and 7-day arteries, respectively. Histamine, norepinephrine, and serotonin constricted cultured arteries, although responses to histamine and norepinephrine diminished by 30-50% after 5 days in culture. The relaxant effect of acetylcholine was not maintained in cultured arteries. Sodium nitroprusside, however, effectively relaxed arteries cultured for 2-7 days. The data indicate that with the culture model described, KATP channels in arterial smooth muscle remained functional and contractile responses in arterial segments were maintained for up to 7 days. These results suggest that this approach can be used to study either long-term regulation of KATP channels or the role of this channel type in growth responses.

scholarly journals Arecoline Hydrobromide Enhances Jejunum Smooth Muscle Contractility via Voltage-Dependent Potassium Channels in W/Wv Mice

2021 ◽  
pp. 437-446
Author(s):  
Q CHEN ◽  
Z JIANG ◽  
J ZHANG ◽  
L CAO ◽  
Z CHEN
Keyword(s):  
Smooth Muscle ◽  
Potassium Channels ◽  
Smooth Muscle Cells ◽  
Isometric Force ◽  
Muscle Cells ◽  
Resting Membrane Potential ◽  
Dependent Manner ◽  
Excitatory Effect ◽  
Patch Clamp Technique ◽  
Voltage Dependent

Gastrointestinal motility was disturbed in W/Wv, which were lacking of interstitial cells of Cajal (ICC). In this study, we have investigated the role of arecoline hydrobromide (AH) on smooth muscle motility in the jejunum of W/Wv and wild-type (WT) mice. The jejunum tension was recorded by an isometric force transducer. Intracellular recording was used to identify whether AH affects slow wave and resting membrane potential (RMP) in vitro. The whole-cell patch clamp technique was used to explore the effects of AH on voltage-dependent potassium channels for jejunum smooth muscle cells. AH enhanced W/Wv and WT jejunum contractility in a dose-dependent manner. Atropine and nicardipine completely blocked the excitatory effect of AH in both W/Wv and WT. TEA did not reduce the effect of AH in WT, but was sufficient to block the excitatory effect of AH in W/Wv. AH significantly depolarized the RMP of jejunum cells in W/Wv and WT. After pretreatment with TEA, the RMP of jejunum cells indicated depolarization in W/Wv and WT, but subsequently perfused AH had no additional effect on RMP. AH inhibited the voltage-dependent K+ currents of acutely isolated mouse jejunum smooth muscle cells. Our study demonstrate that AH enhances the contraction activity of jejunum smooth muscle, an effect which is mediated by voltage-dependent potassium channels that acts to enhance the excitability of jejunum smooth muscle cells in mice.

scholarly journals Mechanisms underlying activation of transient BK current in rabbit urethral smooth muscle cells and its modulation by IP3-generating agonists

2013 ◽  
Vol 305 (6) ◽  
pp. C609-C622 ◽  
Author(s):  
Barry D. Kyle ◽  
Eamonn Bradley ◽  
Roddy Large ◽  
Gerard P. Sergeant ◽  
Noel G. McHale ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Action Potentials ◽  
Muscle Cells ◽  
Current Clamp ◽  
Patch Clamp Technique ◽  
Intracellular Ca ◽  
Urethral Smooth Muscle ◽  
Perforated Patch Clamp

We used the perforated patch-clamp technique at 37°C to investigate the mechanisms underlying the activation of a transient large-conductance K+ (tBK) current in rabbit urethral smooth muscle cells. The tBK current required an elevation of intracellular Ca2+, resulting from ryanodine receptor (RyR) activation via Ca2+-induced Ca2+ release, triggered by Ca2+ influx through L-type Ca2+ (CaV) channels. Carbachol inhibited tBK current by reducing Ca2+ influx and Ca2+ release and altered the shape of spike complexes recorded under current-clamp conditions. The tBK currents were blocked by iberiotoxin and penitrem A (300 and 100 nM, respectively) and were also inhibited when external Ca2+ was removed or the CaV channel inhibitors nifedipine (10 μM) and Cd2+ (100 μM) were applied. The tBK current was inhibited by caffeine (10 mM), ryanodine (30 μM), and tetracaine (100 μM), suggesting that RyR-mediated Ca2+ release contributed to the activation of the tBK current. When IP3 receptors (IP3Rs) were blocked with 2-aminoethoxydiphenyl borate (2-APB, 100 μM), the amplitude of the tBK current was not reduced. However, when Ca2+ release via IP3Rs was evoked with phenylephrine (1 μM) or carbachol (1 μM), the tBK current was inhibited. The effect of carbachol was abolished when IP3Rs were blocked with 2-APB or by inhibition of muscarinic receptors with the M3 receptor antagonist 4-diphenylacetoxy- N-methylpiperidine methiodide (1 μM). Under current-clamp conditions, bursts of action potentials could be evoked with depolarizing current injection. Carbachol reduced the number and amplitude of spikes in each burst, and these effects were reduced in the presence of 2-APB. In the presence of ryanodine, the number and amplitude of spikes were also reduced, and carbachol was without further effect. These data suggest that IP3-generating agonists can modulate the electrical activity of rabbit urethral smooth muscle cells and may contribute to the effects of neurotransmitters on urethral tone.

scholarly journals Magnesium induced vascular relaxation and role of Calcium-dependent K+ Channels

2013 ◽  
Vol 1 (1) ◽  
pp. 9-13
Author(s):  
K Upadhyay-Dhungel ◽  
CJ Kim ◽  
A Dhungel
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Channel Blocker ◽  
Muscle Cells ◽  
K Channel ◽  
Vascular Relaxation ◽  
Patch Clamp Technique ◽  
Calcium Dependent ◽  
K Currents

Background and objectives: Magnesium is established as a neuro-protective agent and now also known as a vasodilator. It has been known for treating vasospasm following subarachnoid hemorrhage. However, its action mechanism in cerebral vascular relaxation is not clear. Potassium channels play a pivotal role in the relaxation of smooth muscle cells. To investigate their role in magnesium-induced relaxation of basilar smooth muscle cells, we examined the effect of magnesium on potassium channels using the patch clamp technique on cells from rabbit basilar artery. Material and Methods: Fresh smooth muscle cells were isolated from the basilar artery by enzyme treatment. Whole cell current recording was done using patch-clamp technique. Appropriate bath solution was used to have potassium current. The effect of Magnesium was observed and to identify the potassium (K+) channel involved in the magnesium-induced currents, different potassium channel blockers were used. Results: Magnesium increased the step pulse-induced outward K+ currents by more than fortyfive percent over control level (p<0.01). The outward K+ current was decreased significantly by application of tetraethylammonium, a non-specific K+ channel blocker, and by iberiotoxin, a largeconductance Ca2+-activated K+ (BKCa) channel blocker, but was not inhibited by glibenclamide an ATP-sensitive K+ (KATP) channel blocker. Magnesium failed to increase the outward K+ currents in the presence of IBX. Conclusion: These results demonstrate that calcium dependent pottassium (BKCa) channels has role in magnesium induced vascular relaxation in rabbit basilar smooth muscle cells and needs to be worked out for human. DOI: http://dx.doi.org/10.3126/jmcjms.v1i1.7880 Janaki Medical College Journal of Medical Sciences (2013) Vol. 1 (1):9-13

Volatile anesthetics inhibit voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells

1995 ◽  
Vol 268 (2) ◽  
pp. L187-L191 ◽  
Author(s):  
M. Yamakage ◽  
C. A. Hirshman ◽  
T. L. Croxton
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Volatile Anesthetics ◽  
Inhibitory Effect ◽  
Tracheal Smooth Muscle ◽  
Lipid Phase ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp

The relaxation of airway smooth muscle by volatile anesthetics is associated with a decreased concentration of intracellular free Ca2+. We hypothesized that inhibition of the entry of extracellular Ca2+ contributes to the relaxation. We therefore examined the effects of halothane, isoflurane, and sevoflurane on macroscopic voltage-activated Ca2+ currents (ICa) in porcine tracheal smooth muscle cells, using the whole cell patch-clamp technique. All three volatile anesthetics significantly inhibited ICa in a dose-dependent manner with no apparent shift in the voltage dependence of induced ICa. The order of inhibitory potencies for ICa was halothane < isoflurane < sevoflurane. When data were plotted as a function of the estimated anesthetic concentrations in the lipid phase, the potencies for inhibition of ICa by the three anesthetics were indistinguishable. We conclude that volatile anesthetics have an inhibitory effect on ICa of porcine tracheal smooth muscle cells at clinically relevant concentrations and that the inhibitory potencies of volatile anesthetics on ICa are closely related to their lipid-phase solubilities.

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