scholarly journals Extracellular acidosis activates ASIC-like channels in freshly isolated cerebral artery smooth muscle cells

2010 ◽  
Vol 298 (5) ◽  
pp. C1198-C1208 ◽  
Author(s):  
Wen-Shuo Chung ◽  
Jerry M. Farley ◽  
Alyssa Swenson ◽  
John M. Barnard ◽  
Gina Hamilton ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cerebral Artery ◽  
Reversal Potential ◽  
Muscle Cells ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Extracellular Acidosis ◽  
Inward Currents ◽  
Induced Currents

Recent studies suggest that certain acid-sensing ion channels (ASIC) are expressed in vascular smooth muscle cells (VSMCs) and are required for VSMC functions. However, electrophysiological evidence of ASIC channels in VSMCs is lacking. The purpose of this study was to test the hypothesis that isolated cerebral artery VSMCs express ASIC-like channels. To address this hypothesis, we used RT-PCR, Western blotting, immunolabeling, and conventional whole cell patch-clamp technique. We found extracellular H+-induced inward currents in 46% of cells tested ( n = 58 of 126 VSMCs, pH 6.5–5.0). The percentage of responsive cells and the current amplitude increased as the external H+ concentration increased (pH6.0, n = 28/65 VSMCs responsive, mean current density = 8.1 ± 1.2 pA/pF). Extracellular acidosis (pH6.0) shifted the whole cell reversal potential toward the Nernst potential of Na+ ( n = 6) and substitution of extracellular Na+ by N-methyl-d-glucamine abolished the inward current ( n = 6), indicating that Na+ is a major charge carrier. The broad-spectrum ASIC blocker amiloride (20 μM) inhibited proton-induced currents to 16.5 ± 8.7% of control ( n = 6, pH6.0). Psalmotoxin 1 (PcTx1), an ASIC1a inhibitor and ASIC1b activator, had mixed effects: PcTx1 either 1) abolished H+-induced currents (11% of VSMCs, 5/45), 2) enhanced or promoted activation of H+-induced currents (76%, 34/45), or 3) failed to promote H+ activation in nonresponsive VSMCs (13%, 6/45). These findings suggest that freshly dissociated cerebral artery VSMCs express ASIC-like channels, which are predominantly formed by ASIC1b.

Voltage-dependent calcium channel current in isolated gallbladder smooth muscle cells of guinea pig

1993 ◽  
Vol 264 (6) ◽  
pp. G1066-G1076 ◽  
Author(s):  
T. Shimada
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Time Course ◽  
Reversal Potential ◽  
High Sensitivity ◽  
Muscle Cells ◽  
Patch Clamp Technique ◽  
Decay Component ◽  
Voltage Dependent

The voltage-dependent Ca2+ current was studied in enzymatically dispersed guinea pig gallbladder smooth muscle cells using the whole cell patch-clamp technique. Depolarizing voltage (V) steps induced an inward current (I) that was carried by Ca2+. The threshold potential was -40 to -30 mV, the maximal current was observed at +10 to +20 mV, and the reversal potential was around +80 mV. I-V curves obtained with holding potentials of -80 and -40 mV were not significantly different. This current had a high sensitivity to dihydropyridine drugs, and the Ba2+ or Sr2+ current was larger than the Ca2+ current. Activation was accelerated by increasing the membrane potential. In general, the time course of decay was well fitted by the sum of two exponentials, but consideration of a third (ultra-slow) decay component was also necessary when the current generated by a 2-s command pulse was analyzed. Superimposition of activation and inactivation curves showed the presence of a significant window current. Carbachol suppressed the Ca2+ current only when the pipette contained a low concentration of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. These results show that the L-type Ca2+ current is dominant in gallbladder smooth muscle cells and may contribute to excitation-contraction coupling.

Nitric oxide suppresses a Ca2+-stimulated Cl− current in smooth muscle cells of opossum esophagus

1998 ◽  
Vol 274 (5) ◽  
pp. G886-G890 ◽  
Author(s):  
Yong Zhang ◽  
Fivos Vogalis ◽  
Raj K. Goyal
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Smooth Muscles ◽  
Reversal Potential ◽  
Circular Muscle ◽  
Muscle Cells ◽  
Patch Clamp Technique ◽  
No Donor ◽  
Pipette Solution

Nitric oxide (NO) hyperpolarizes visceral smooth muscles. Using the patch-clamp technique, we investigated the possibility that NO-mediated hyperpolarization in the circular muscle of opossum esophagus results from the suppression of a Ca2+-stimulated Cl− current. Smooth muscle cells were dissociated from the circular layer and bathed in high-K+Ca2+-EGTA-buffered solution. Macroscopic ramp currents were recorded from cell-attached patches. Contaminating K+-channel currents were blocked with tetrapentylammonium chloride (200 μM) added to all solutions. Raising bath Ca2+concentration above 150 nM in the presence of A-23187 (10 μM) activated a leak current ( I L-Ca) with an EC50 of 1.2 μM at −100 mV. The reversal potential ( E rev) of I L-Ca (−8.5 ± 1.8 mV, n = 8) was significantly different ( P < 0.05) from E rev of the background current (+4.2 ± 1.2 mV, n = 8). Equimolar substitution of 135 mM Cl− in the pipette solution with gluconate significantly shifted E rev of I L-Ca to +16.6 ± 3.4 mV ( n = 4) ( P < 0.05 compared with background), whereas replacement of total Na+with Tris+ suppressed I L-Ca but did not affect E rev(−15 ± 3 mV, n = 3; P > 0.05). I L-Ca was inhibited by DIDS (500 μM). Diethylenetriamine-NO adduct (200 μM), a NO• donor, and 8-bromo-cGMP (200 μM) suppressed I L-Ca by 59 ± 15% ( n = 5) and 62 ± 21% ( n = 4) at −100 mV, respectively. We conclude that in opossum esophageal smooth muscle NO-mediated hyperpolarization may be produced by suppression of a Ca2+-stimulated Cl−-permeable conductance via formation of cGMP.

Nitric oxide modulates a calcium-activated potassium current in muscle cells from opossum esophagus

1995 ◽  
Vol 269 (4) ◽  
pp. G606-G612 ◽  
Author(s):  
J. A. Murray ◽  
E. F. Shibata ◽  
T. L. Buresh ◽  
H. Picken ◽  
B. W. O'Meara ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Protein Kinase ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Protein Kinase G ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Circular Smooth Muscle ◽  
K Current

Nitric oxide mediates nerve-induced hyperpolarization of circular smooth muscle of the esophagus. Two mechanisms are proposed to explain this hyperpolarization: an increase in K+ current or a decrease in Cl- current. These studies test the hypothesis that nitric oxide increases a K+ current in esophageal smooth muscle. Three outward K+ currents are present in circular smooth muscle cells from the opossum esophagus. One current is a Ca(2+)-activated K+ current (IKCa2+). This current is inhibited by charybdotoxin. Whole cell currents were recorded from isolated opossum esophageal smooth muscle cells using the whole cell patch-clamp technique. These studies showed that IKCa2+ is activated at potentials more positive than -30 mV. Bath application of S-nitroso-L-cysteine increased IKCa2+ by 50% above control levels throughout the entire activation range of potentials. The enhanced current was reversible on washout. Either charybdotoxin, an inhibitor of IKCa2+, or (R)-p-8-(4-chloropenylthio)-guanosine 3',5'-cyclic monophosphorothioate, an inhibitor of protein kinase G, antagonized the increase in outward current induced by S-nitroso-L-cysteine. These data suggest that nitric oxide activates IKCa2+ via the guanosine 3',5'-cyclic monophosphate-protein kinase G signal transduction pathway.

Calcium channel currents in isolated smooth muscle cells from human bronchus

1989 ◽  
Vol 66 (4) ◽  
pp. 1706-1714 ◽  
Author(s):  
R. Marthan ◽  
C. Martin ◽  
T. Amedee ◽  
J. Mironneau
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Electrophysiological Study ◽  
Reversal Potential ◽  
External Solution ◽  
Muscle Cells ◽  
Patch Clamp Technique ◽  
Inward Current ◽  
Human Bronchus ◽  
A Charge

An electrophysiological study was carried out on smooth muscle cells that were enzymatically dissociated from bundles of muscle fibers dissected out of human bronchi obtained at thoracotomy. These cells that retain the contractile properties of intact bundles were voltage-clamped by means of the whole-cell patch-clamp technique. Upon voltage steps from a holding potential of -60 mV to more positive levels, the initial inward current was followed by large outward currents that inactivated slowly. These were subsequently reduced by substituting Cs+ for K+ in the internal solution and by using Ba2+ instead of Ca2+ as a charge carrier in the external solution. Under these conditions, the inward current did not completely inactivate in the course of 300-ms voltage steps. Inward current measured after leak subtraction was activated at a membrane potential of -25.8 +/- 5 mV, was maximum at +18 +/- 4 mV, and had an apparent reversal potential of +52.5 +/- 5.5 mV (n = 5). The potential at which steady-state inactivation was half-maximum was -28 mV (n = 5). This inward current was identified as a calcium current on the following basis: 1) it was not altered by 10 microM tetrodotoxin (TTX) or by lowering to 10 mM external Na+ concentration; 2) it was blocked by 2.5 mM Co2+ or 1 microM PN 200–110; 3) it was enhanced by 1 microM BAY K 8644, which in addition suppressed the PN 200–110 blockade.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-sensitive K+ currents in cerebral arterial smooth muscle: pharmacological and hormonal modulation

1995 ◽  
Vol 269 (5) ◽  
pp. H1634-H1640 ◽  
Author(s):  
T. Kleppisch ◽  
M. T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cerebral Arteries ◽  
Katp Channels ◽  
Muscle Cells ◽  
Patch Clamp Technique ◽  
Calcitonin Gene ◽  
Hormonal Modulation ◽  
Calcium Activated Potassium Channels ◽  
Induced Currents

Calcitonin gene-related peptide (CGRP), hypoxia, and synthetic activators of ATP-sensitive potassium (KATP) channels (e.g., pinacidil and levcromakalim) cause dilation of cerebral arteries that are attenuated by the KATP channel inhibitor glibenclamide. We have identified and characterized KATP currents in smooth muscle cells isolated from rabbit cerebral arteries, using the whole cell configuration of the patch-clamp technique. Pinacidil (10 microM) and levcromakalim (10 microM) increased glibenclamide-sensitive currents about sixfold in cells dialyzed with 0.1 mM ATP. Glibenclamide-sensitive currents in the presence of pinacidil were potassium selective, voltage independent, and reduced about threefold by elevating intracellular ATP from 0.1 to 3.0 mM. External tetraethylammonium and 4-aminopyridine at millimolar concentrations reduced pinacidil-induced currents, whereas iberiotoxin, a blocker of calcium-activated potassium channels, had no effect. The vasoconstrictors serotonin and histamine also inhibited pinacidil-induced currents. The vasodilators CGRP and adenosine, in contrast, increased glibenclamide-sensitive potassium currents. We conclude that cerebral artery smooth muscle cells have KATP channels that are regulated by endogenous vasoconstrictors and vasodilators. We propose that these channels are involved in the dilation of cerebral arteries to CGRP and synthetic vasodilators.

Ionic currents and endothelin signaling in smooth muscle cells from rat renal resistance arteries

1994 ◽  
Vol 266 (2) ◽  
pp. F325-F341 ◽  
Author(s):  
D. V. Gordienko ◽  
C. Clausen ◽  
M. S. Goligorsky
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Reversal Potential ◽  
Muscle Cells ◽  
Membrane Depolarization ◽  
Disulfonic Acid ◽  
Patch Clamp Technique ◽  
Voltage Range ◽  
Voltage Gated ◽  
Ca2 Channels

The repertoire of ionic channels expressed in myocytes freshly isolated from microdissected interlobar and arcuate arteries of rat kidney and their integrative behavior in response to endothelin-1 (ET-1) were studied by identification and characterization of major whole cell current components using patch-clamp technique. In renal microvascular smooth muscle cells (RMSMC) dialyzed with K(+)-containing solution, rapidly inactivating (Ito) and sustained outward K+ currents were identified. Voltage-dependent Ito was categorized as "A" current based on its kinetics, sensitivity to 4-aminopyridine (4-AP), and refractoriness to tetraethylammonium (TEA+). Ca(2+)-activated component of K+ current was completely blocked by 10 mM TEA+, whereas 5 mM 4-AP did not affect this current. Maximal Ca2+ current (ICa) recorded in Cs(+)-loaded RMSMC reached 250 pA when cells were bathed in a solution with 2.5 mM Ca2+. Two patterns of ICa differing in kinetics, voltage range of activation and inactivation, and sensitivity to nifedipine were identified as T and L currents. Ca(2+)-dependent current component showing reversal potential near Cl- current (ECl) and sensitivity to blocking action of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was identified as Ca(2+)-activated ECl. Activation of RMSMC with ET-1 (1-10 nM) induced elevation of [Ca2+]i and subsequent activation of Ca(2+)-activated ICl, which led to membrane depolarization sufficient to activate voltage-gated Ca2+ channels. ET-1-evoked transient reduction of ICa carried through voltage-gated Ca2+ channels was followed by augmentation of L-type ICa. ET-1-induced mobilization of intracellular Ca2+, accompanied by membrane depolarization, resulted in activation of Ca(2+)-dependent K+ channels, which can play the role of a feedback element terminating ET-1-induced membrane depolarization.

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.

Inward rectifier K+ currents in smooth muscle cells from rat resistance-sized cerebral arteries

1993 ◽  
Vol 265 (5) ◽  
pp. C1363-C1370 ◽  
Author(s):  
J. M. Quayle ◽  
J. G. McCarron ◽  
J. E. Brayden ◽  
M. T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cerebral Arteries ◽  
Muscle Cells ◽  
Inward Rectifier ◽  
Equilibrium Potential ◽  
Membrane Hyperpolarization ◽  
Whole Cell ◽  
Inward Currents ◽  
K Currents

Inward rectifier K+ channels have been implicated in the control of membrane potential and external K(+)-induced dilations of small cerebral arteries. In the present study, whole cell K+ currents through the inward rectifier K+ channel were measured in single smooth muscle cells isolated from the posterior cerebral artery of Wistar-Kyoto rats. The whole cell K+ current-voltage relationship showed inward rectification. Inward currents were recorded negative to the K+ equilibrium potential, whereas outward currents were small. When extracellular K+ was elevated, the zero current potential shifted to the new K+ equilibrium potential, and the conductance of the inward current increased. Inward currents were reduced by external barium or cesium. Inhibition by barium and cesium increased with membrane hyperpolarization. The half-inhibition constant for barium was 2.2 microM at -60 mV, increasing e-fold for a 23-mV depolarization. We provide the first direct measurements of inward rectifier K+ currents in single smooth muscle cells and show that external barium ions are effective blockers of these currents.

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