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.

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.

scholarly journals Multiple muscarinic pathways mediate the suppression of voltage-gated Ca2+ channels in mouse intestinal smooth muscle cells

2009 ◽  
Vol 158 (8) ◽  
pp. 1874-1883 ◽  
Author(s):  
Yasuyuki Tanahashi ◽  
Toshihiro Unno ◽  
Hayato Matsuyama ◽  
Toshiaki Ishii ◽  
Masahisa Yamada ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Intestinal Smooth Muscle ◽  
Voltage Gated ◽  
Ca2 Channels

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.

Increased intracellular Na+ augments mobilization of Ca2+ from SR in vascular smooth muscle cells

1994 ◽  
Vol 266 (1) ◽  
pp. C311-C317 ◽  
Author(s):  
M. L. Borin ◽  
R. M. Tribe ◽  
M. P. Blaustein
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Tetraacetic Acid ◽  
Free Medium ◽  
Atpase Inhibitor ◽  
Voltage Gated ◽  
Ca2 Channels

The effect of a rise in intracellular Na+ concentration ([Na+]cyt) on the amount of Ca2+ in intracellular stores was studied in vascular smooth muscle cells from the A7r5 line. The relative amount of stored Ca2+ was estimated in fura 2-loaded cells by the rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) evoked by Ca2+ release from the sarcoplasmic reticulum (SR). To improve the detection of released Ca2+, extrusion of Ca2+ from the cytosol was minimized by using nominally Na+/Ca(2+)-free medium containing 0.5 mM La3+ [for vasoconstrictor experiments, the medium contained 0.5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and no La3+]. Ca2+ release was triggered by thapsigargin (TG), an SR Ca(2+)-ATPase inhibitor, and by the vasoconstrictors arginine vasopressin (AVP) and serotonin (5-HT). Incubation with 1-3 mM ouabain for 20 min, which raises [Na+]cyt from 4.4 to 9.0 mM, increased "resting" [Ca2+]cyt only slightly (from 87 to 122 nM). However, ouabain greatly augmented the release of Ca2+ evoked by TG [from 639 nM (control) to 1,021 nM], by AVP (from 993 to 1,597 nM), and by 5-HT (from 559 to 1,486 nM). Ouabain-induced augmentation of TG-evoked Ca2+ release was not affected by 10 microM verapamil; this implies that the effect of ouabain was not due to Ca2+ entry through voltage-gated Ca2+ channels. The response to TG was not augmented when ouabain was applied for 20 min in Na(+)-free medium (Na+ replaced by equimolar N-methyl-D-glucamine) to prevent [Na+]cyt from rising.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

scholarly journals A type 2A phosphatase-sensitive phosphorylation site controls modal gating of L-type Ca2+ channels in human vascular smooth-muscle cells

1996 ◽  
Vol 318 (2) ◽  
pp. 513-517 ◽  
Author(s):  
Klaus GROSCHNER ◽  
Klaus SCHUHMANN ◽  
Gottfried MIESKES ◽  
Werner BAUMGARTNER ◽  
Christoph ROMANIN
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Okadaic Acid ◽  
Muscle Cells ◽  
Catalytic Subunit ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Mode 2 ◽  
Modal Gating ◽  
Ca2 Channels

The patch-clamp technique was employed to investigate phosphorylation/dephosphorylation-dependent modulation of L-type Ca2+ channels in smooth-muscle cells isolated from human umbilical vein. Okadaic acid, an inhibitor of phosphoprotein phosphatases type 1 (PP1) and 2A (PP2A), increased the probability of channels being in the open state (Po) in intact cells. This increase in Po was due mainly to promotion of long-lasting channel openings, i.e. promotion of ‘mode 2’ gating behaviour. Exposure of the cytoplasmic side of excised patches of membrane to the purified catalytic subunit of PP2A (PP2Ac) resulted in the opposite modulation of channel function. PP2Ac (0.2 unit/ml) reduced the Po of Ca2+ channels mainly via suppression of ‘mode 2’ gating. This effect of PP2Ac was completely prevented by 1 µM okadaic acid. The catalytic subunit of PP1 (0.2 unit/ml), however, barely affected channel activity. Our results provide evidence for a PP2A-sensitive regulatory site that controls modal gating of L-type Ca2+ channels in smooth muscle.

The cardiac sodium current Nav1.5 is functionally expressed in rabbit bronchial smooth muscle cells

2013 ◽  
Vol 305 (4) ◽  
pp. C427-C435 ◽  
Author(s):  
E. Bradley ◽  
T. I. Webb ◽  
M. A. Hollywood ◽  
G. P. Sergeant ◽  
N. G. McHale ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Sodium Current ◽  
Single Cells ◽  
Muscle Cells ◽  
Airway Smooth Muscle Cells ◽  
Patch Clamp Technique ◽  
Human Embryonic Kidney Cells ◽  
Fourfold Increase ◽  
Voltage Gated

A collagenase-proteinase mixture was used to isolate airway smooth muscle cells (ASMC) from rabbit bronchi, and membrane currents were recorded using the whole cell patch-clamp technique. Stepping from −100 mV to a test potential of −40 mV evoked a fast voltage-dependent Na+ current, sometimes with an amplitude of several nanoamperes. The current disappeared within 15 min of exposure to papain + DTT ( n = 6). Comparison of the current in ASMC with current mediated by NaV1.5 α-subunits expressed in human embryonic kidney cells revealed similar voltage dependences of activation ( V1/2 = −42 mV for NaV1.5) and sensitivities to TTX (IC50 = 1.1 and 1.2 μM for ASMC and NaV1.5, respectively). The current in ASMC was also blocked by lidocaine (IC50 = 160 μM). Although veratridine, an agonist of voltage-gated Na+ channels, reduced the peak current by 33%, it slowed inactivation, resulting in a fourfold increase in sustained current (measured at 25 ms after onset). In current-clamp mode, veratridine prolonged evoked action potentials from 37 ± 9 to 1,053 ± 410 ms ( n = 8). Primers for NaV1.2–1.9 were used to amplify mRNA from groups of ∼20 isolated ASMC and from whole bronchial tissue by RT-PCR. Transcripts for NaV1.2, NaV1.3, and NaV1.5–1.9 were detected in whole tissue, but only NaV1.2 and NaV1.5 were detected in single cells. We conclude that freshly dispersed rabbit ASMC express a fast voltage-gated Na+ current that is mediated mainly by the NaV1.5 subtype.

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.

Activation of chloride currents in murine portal vein smooth muscle cells by membrane depolarization involves intracellular calcium release

2005 ◽  
Vol 288 (1) ◽  
pp. C122-C131 ◽  
Author(s):  
Sohag N. Saleh ◽  
Iain A. Greenwood
Keyword(s):  
Smooth Muscle ◽  
Portal Vein ◽  
Smooth Muscle Cells ◽  
Calcium Release ◽  
Reversal Potential ◽  
Muscle Cells ◽  
Membrane Depolarization ◽  
Isometric Tension ◽  
Channel Blockers ◽  
Atpase Inhibitor

The present study describes the first characterization of Ca2+-activated Cl− currents ( IClCa) in single smooth muscle cells from a murine vascular preparation (portal veins). IClCa was recorded using the perforated patch version of the whole cell voltage-clamp technique and was evoked using membrane depolarization. Generation of IClCa relied on Ca2+ entry through dihydropyridine-sensitive Ca2+ channels because IClCa was abolished by 1 μM nicardipine and enhanced by raising external Ca2+ concentration or by application of BAY K 8644. IClCa was characterized by the sensitivity to Cl− channel blockers and the effect of altering the external anion on reversal potential. Activation of IClCa after membrane depolarization was dependent on Ca2+ release from intracellular stores. Thus the amplitude of IClCa was diminished by the SR-ATPase inhibitor cyclopiazonic acid, the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate (2-APB), and the ryanodine receptor blocker tetracaine. The degree of inhibition produced by the application of 2-APB and tetracaine together was significantly greater than the effect of each agent applied alone. In current-clamp mode, injection of depolarizing current elicited a biphasic action potential, with the later depolarization being sensitive to niflumic acid (NFA; 10 μM). In isometric tension recordings, NFA inhibited spontaneous contractions. These data support a role for this conductance in portal vein excitability.

Evidence for presence of ATP-sensitive K+ channels in rat colonic smooth muscle cells

1998 ◽  
Vol 76 (12) ◽  
pp. 1166-1170 ◽  
Author(s):  
Lídia Plujà ◽  
Hisashi Yokoshiki ◽  
Nicholas Sperelakis
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Reversal Potential ◽  
Cell Voltage ◽  
Muscle Cells ◽  
Equilibrium Potential ◽  
Rat Colon ◽  
Sulfonylurea Receptor ◽  
Patch Clamp Technique

Coexpression of sulfonylurea receptor (SUR) and inward-rectifying K+ channel (Kir6.1 or 6.2) subunit yields ATP-sensitive K+ (KATP) channels. Three subtypes of SUR have been cloned: pancreatic (SUR1), cardiac (SUR2A), and vascular smooth muscle (SUR2B). The distinct responses to K+ channel openers (KCOs) produced in different tissues may depend on the SUR isoform of KATP channel. Therefore, we investigated the effects of pinacidil and diazoxide, two KCOs, on KATP currents in intestinal smooth muscle cells of the rat colon (circular layer) using whole-cell voltage clamp. Pinacidil stimulated a time-independent K+ current evoked by various test potentials from a holding potential of -70 mV. The reversal potential of the stimulated current was about -75 mV, which is close to the equilibrium potential for K+ (EK). Both pinacidil and diazoxide dose-dependently stimulated K+ currents (evoked by ramp pulses), with EC50 values of 1.3 and 34.2 µM, respectively. The stimulated current was completely reversed by glybenclamide (3 µM). Since the EC50 values are close to those reported for vascular smooth muscle (VSM) cells, the SUR subtype may be similar to that in VSM cells, and could form the functional KATP channel in rat colonic smooth muscle cells.Key words: ATP-sensitive K+ channels, rat colonic smooth muscle cells, pinacidil, diazoxide, patch-clamp technique.

Sign in / Sign up

Export Citation Format

Share Document