G proteins mediate suppression of Ca2+-activated K current by acetylcholine in smooth muscle cells

1989 ◽  
Vol 257 (3) ◽  
pp. C596-C600 ◽  
Author(s):  
W. C. Cole ◽  
K. M. Sanders
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Outward Current ◽  
Muscle Cells ◽  
Pipette Solution ◽  
Whole Cell ◽  
K Channels ◽  
K Current

The role of G proteins in cholinergic suppression of Ca2+-activated K current was studied in isolated canine colonic myocytes with the whole cell voltage-clamp technique. Acetylcholine (ACh; 10.0 microM) caused a 64 +/- 2.4% depression in the Ca2+-dependent component of the outward current evoked at potentials between -45 and -15 mV when GTP (0.1 microM) was included in the pipette-filling solution. This effect was reversed within 2-4 min on washout of ACh. Without GTP in the filling solution, ACh caused a 15 +/- 2.5% depression in outward current in 60% of the cells tested. When the non-hydrolyzable GTP analogues, GTP gamma S (0.1 mM) or 5'-guanylylimidodiphosphate (GppNHp; 0.1 mM) were used, the decrease in outward current was greater (85 +/- 4.2 and 78 +/- 6.5%, respectively), and it was not reversed on withdrawal of ACh. Dialysis of the cell interior with pipette solution containing pertussis toxin (1 ng/ml) for 30 min had no effect on the whole cell currents evoked on depolarization, but it abolished the effect of ACh on Ca2+-dependent outward current. These data suggest that coupling of muscarinic receptors to the inhibition of Ca2+-activated K channels is mediated by pertussis toxin-sensitive G proteins in colonic smooth muscle cells. G protein-mediated inhibition is distinctly different from the opening of muscarinic-regulated K channels in other cell types.

Src-Dependence and Pertussis-Toxin Sensitivity of Urokinase Receptor-Dependent Chemotaxis and Cytoskeleton Reorganization in Rat Smooth Muscle Cells

Blood ◽  
1999 ◽  
Vol 94 (2) ◽  
pp. 649-662 ◽  
Author(s):  
Bernard Degryse ◽  
Massimo Resnati ◽  
Shafaat A. Rabbani ◽  
Antonello Villa ◽  
Francesca Fazioli ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Cell Shape ◽  
Muscle Cells ◽  
Soluble Form ◽  
Integrin Subunit ◽  
Cytoskeleton Reorganization

The catalytically inactive precursor of urokinase-type plasminogen activator (pro-u-PA) induced a chemotactic response in rat smooth muscle cells (RSMC) through binding to the membrane receptor of urokinase (u-PA receptor [u-PAR]). A soluble form of u-PAR activated by chymotrypsin cleavage as well as a peptide located between domain 1 and 2 of u-PAR reproduced the effect of pro-u-PA on cell migration. The chemotactic pro-u-PA effect correlates with a dramatic reorganization of actin cytoskeleton, of adhesion plaques, and with major cell shape changes in RSMC. Pro-u-PA induced a decrease in stress fiber content, membrane ruffling, actin ring formation, and disruption leading to the characteristic elongated cell shape of motile cells with an actin semi-ring located close to the leading edge of cells. u-PAR effects on both chemotaxis and cytoskeleton were sensitive to pertussis toxin and, hence, possibly require G proteins. u-PAR effects are accompanied by a relocation of u-PAR, vitronectin receptor (VNR) vβ3, β1 integrin subunit, and Src tyrosine kinase to the leading membrane of migrating cells. In conclusion, our data show that pro-u-PA, via binding to u-PAR, controls a signaling pathway, regulated by tyrosine kinases and possibly G proteins, leading to cell cytoskeleton reorganization and cell migration.

Calcium and potassium currents in canine gastric smooth muscle cells

1992 ◽  
Vol 262 (5) ◽  
pp. G859-G867 ◽  
Author(s):  
S. M. Sims
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Outward Current ◽  
Muscle Cells ◽  
Current Evidence ◽  
Equilibrium Potential ◽  
Inward Current ◽  
K Current ◽  
Gastric Corpus ◽  
Ca2 Channels

Membrane ionic currents were recorded in single smooth muscle cells dissociated from circular muscle of dog stomach (corpus region). When studied under voltage clamp with K+ in the patch electrode, depolarization to potentials more positive than -40 mV, from a holding potential of -70 or -80 mV, evoked transient inward current followed by outward current. Evidence that the outward current was due to K+ came from analysis of deactivation tail currents, which reversed direction close to the K+ equilibrium potential. In addition, the outward current was reduced by tetraethylammonium (TEA, 1-5 mM) applied to the external surface of cells. The Ca(2+)-channel blocker Cd2+ blocked the inward current and also reduced outward current, suggesting Ca(2+)-activated K+ current contributed to the outward current. The voltage-activated inward current was studied in isolation with Cs+ and TEA in the recording electrode to block K+ current. In standard bathing solution containing 2.5 mM Ca2+, the inward current activated between -50 and -40 mV, with peak inward current at +10 mV. The depolarization-activated inward current was blocked by nifedipine and enhanced by BAY K 8644, providing evidence that it was Ca2+ current. The Ca2+ current showed transient and sustained components, both of which showed similar voltage activation and inactivation ranges. The half-inactivation potential was approximately -37 mV. These results provide evidence that smooth muscle cells from the canine gastric corpus possess K+ and Ca2+ channels. Based on the voltage dependence of activation and inactivation and sensitivity to dihydropyridines, L-type Ca2+ channels predominate in canine gastric corpus smooth muscle.

Membrane currents and cholinergic regulation of K+ current in esophageal smooth muscle cells

1990 ◽  
Vol 258 (5) ◽  
pp. G794-G802 ◽  
Author(s):  
S. M. Sims ◽  
M. B. Vivaudou ◽  
C. Hillemeier ◽  
P. Biancani ◽  
J. V. Walsh ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Membrane Currents ◽  
Equilibrium Potential ◽  
Whole Cell ◽  
Inward Current ◽  
Whole Cell Recording ◽  
Cell Recording ◽  
K Current

The tight-seal whole cell recording technique with patch pipettes was used to study membrane currents of smooth muscle cells freshly dissociated from the esophagus of cats. Under voltage clamp with K+ in the pipette, depolarizing commands elicited an initial inward current followed by a transient outward current that peaked and then declined to reveal spontaneous outward currents (SOCs). SOCs were evident at -60 mV and more positive potentials. The reversal of SOCs at the K+ equilibrium potential and their suppression by tetraethylammonium chloride lead to the conclusion that they represent the activity of K+ channels. Acetylcholine (ACh) caused reversible contraction of these cells and had two successive effects on membrane currents, causing transient activation of K+ current followed by suppression of SOCs. Both of these effects were blocked by atropine. Consistent with these observations, in current clamp, ACh caused a transient hyperpolarization followed by depolarization. The inward current activated by depolarization was blocked by external Cd2+, consistent with the inward current being a voltage-activated calcium current. Two types of Ca2+ current could be distinguished on the basis of voltage-activation range, time course of inactivation and "run-down" during whole cell recording.

Whole cell current and membrane potential regulation by a human smooth muscle mechanosensitive calcium channel

2000 ◽  
Vol 279 (6) ◽  
pp. G1155-G1161 ◽  
Author(s):  
Adrian N. Holm ◽  
Adam Rich ◽  
Michael G. Sarr ◽  
Gianrico Farrugia
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Smooth Muscle Cells ◽  
Outward Current ◽  
Muscle Cells ◽  
Membrane Hyperpolarization ◽  
Whole Cell ◽  
Circular Smooth Muscle ◽  
Channel Current ◽  
Cell Current

Mechanotransduction is required for a wide variety of biological functions. The aim of this study was to determine the effect of activation of a mechanosensitive Ca2+ channel, present in human jejunal circular smooth muscle cells, on whole cell currents and on membrane potential. Currents were recorded using patch-clamp techniques, and perfusion of the bath (10 ml/min, 30 s) was used to mechanoactivate the L-type Ca2+ channel. Perfusion resulted in activation of L-type Ca2+ channels and an increase in outward current from 664 ± 57 to 773 ± 72 pA at +60 mV. Membrane potential hyperpolarized from −42 ± 4 to −50 ± 5 mV. In the presence of nifedipine (10 μM), there was no increase in outward current or change in membrane potential with perfusion. In the presence of charybdotoxin or iberiotoxin, perfusion of the bath did not increase outward current or change membrane potential. A model is proposed in which mechanoactivation of an L-type Ca2+ channel current in human jejunal circular smooth muscle cells results in increased Ca2+ entry and cell contraction. Ca2+ entry activates large-conductance Ca2+-activated K+channels, resulting in membrane hyperpolarization and relaxation.

Ca(2+)-dependent K+ current in arterial smooth muscle cells from aldosterone-salt hypertensive rats

1995 ◽  
Vol 269 (4) ◽  
pp. H1246-H1257 ◽  
Author(s):  
Y. Liu ◽  
A. W. Jones ◽  
M. Sturek
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Outward Current ◽  
Muscle Cells ◽  
Hypertensive Rats ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
Kca Channels ◽  
K Current ◽  
In Cells

Aorta from aldosterone-salt hypertensive rats (AHR) demonstrates an increased basal 42K efflux. We investigated the cellular mechanisms of this alteration by measuring 42K efflux from aortic segments as well as myoplasmic Ca2+ concentration ([Ca2+]m) and K+ current in aortic smooth muscle cells from AHR and normotensive control-salt rats (CSR). Both diltiazem and nisoldipine attenuated but did not normalize the increase in basal 42K efflux in AHR. The resting [Ca2+]m was elevated in cells from AHR (148 +/- 15 vs. 91 +/- 12 nM for CSR, P < 0.05). The rate of Mn2+ quenching under basal conditions was also increased in cells from AHR, and the increase was abolished by Cd2+. However, the resting membrane potential did not differ between CSR and AHR (-49 +/- 5 vs. -50 +/- 4 mV). The steady-state, whole cell K+ current was also increased in cells from AHR. This increase was abolished by charybdotoxin, tetraethylammonium, La3+, and by clamping [Ca2+]m at zero or 100 nM with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The single-channel conductance of the large conductance Ca(2+)-activated, voltage-dependent K+ (KCa) channels was not altered in AHR. Further, 33% of cells from AHR vs. 1% from CSR showed spontaneous transient outward K+ currents, which reflect activation of KCa channels by Ca2+ released from caffeine-sensitive stores. While the acute caffeine-induced [Ca2+]m response was similar between CSR and AHR, the outward current and 42K efflux responses to caffeine were greater in AHR. After continued exposure to caffeine, the basal 42K efflux was attenuated more in AHR than in CSR. Charybdotoxin resulted in a greater depolarization in AHR cells than in CSR cells (9.8 +/- 2.2 vs. 3.5 +/- 1.6 mV, P < 0.05). These results indicate that the increases in both 42K efflux and K+ current reflect an increased activity of KCa channels that is associated with an increased Ca2+ influx and resting [Ca2+]m and altered Ca2+ handling by the sarcoplasmic reticulum in aortic smooth muscle cells from AHR.

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.

Protein kinase C inhibits delayed rectifier K+ current in rabbit vascular smooth muscle cells

1996 ◽  
Vol 271 (1) ◽  
pp. H109-H119 ◽  
Author(s):  
E. A. Aiello ◽  
O. Clement-Chomienne ◽  
D. P. Sontag ◽  
M. P. Walsh ◽  
W. C. Cole
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Outward Current ◽  
Muscle Cells ◽  
Delayed Rectifier ◽  
Whole Cell ◽  
Kinase C

The effect of protein kinase C (PKC) activation on 4-aminopyridine (4-AP)-sensitive delayed rectifier current (IdK) was studied in isolated rabbit portal vein smooth muscle cells by use of standard whole cell voltage clamp. The effects of the phorbol ester, 4 beta-phorbol 12,13-dibutyrate (PdBu, 100 nM) and diacylglycerol analogues, 1,2-dioctanoyl-sn-glycerol (1,2-diC8, 10 microM) and 1,3-dioctanoyl-sn-glycerol (1,3-diC8, 10 microM), on macroscopic whole cell IdK were assessed in myocytes dialyzed with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and 5 mM ATP (20-22 degrees C). Activation of PKC by 1,2-diC8 or PdBu caused a decline in IdK that was reversed with washout of drug. 1,2-diC8 had no effect on outward current present after exposure to 4-AP (20 mM). The inactive analogue, 1,3-diC8, did not affect IdK, but subsequent exposure to the active analogue, 1,2-diC8, caused a marked depression of the current. The inhibition of IdK by 1,2-diC8 was significantly reduced by intracellular dialysis with the inhibitors of PKC, chelerythrine (50 microM) and calphostin C (1 microM). Substitution of extracellular Ca2+ with Mg2+ in the presence of 10 mM intracellular BAPTA did not affect the suppression of IdK by 1,2-diC8, indicating the involvement of a Ca(2+)-independent isoform of PKC. This study suggests a novel signal transduction mechanism for inhibition of 4-AP-sensitive IdK involving a phosphotransferase reaction catalyzed by PKC in vascular smooth muscle myocytes.

ATP-sensitive K+ channels regulate resting potential of pulmonary arterial smooth muscle cells

1992 ◽  
Vol 262 (3) ◽  
pp. H916-H920 ◽  
Author(s):  
L. H. Clapp ◽  
A. M. Gurney
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Resting Potential ◽  
K Channels ◽  
K Current ◽  
Pulmonary Arterial ◽  
Arterial Smooth Muscle Cells ◽  
Pulmonary Arterial Smooth Muscle

ATP-sensitive K+ (KATP) channels have been proposed to be the target for hyperpolarizing vasodilators. However, the existence of a whole cell KATP current that can regulate membrane potential has not been demonstrated in vascular muscle. Using the patch-clamp technique, we have examined the effects of varying intracellular ATP on membrane potential and currents in isolated rabbit pulmonary arterial smooth muscle cells. With 1 mM ATP in the pipette, cells had a mean resting potential of -55 mV. When ATP was omitted, the resting potential became significantly more hyperpolarized (-70 mV) and the depolarizing response to the KATP-channel blocker, glibenclamide, was potentiated. In contrast, the hyperpolarizing effect of lemakalim was reduced. These hyperpolarized resting potentials were associated with increased activity of a basal, glibenclamide-sensitive time-independent K+ current. Furthermore, flash photolysis of ATP, 3-O-[1(4,5-dimethoxy-2-nitrophenyl)ethyl] ester, disodium salt ("caged ATP") in ATP-depleted cells caused rapid depolarization (less than 1 s) and block of the background K+ current. Our results are consistent with the idea that intracellular ATP can directly modulate the resting potential by inhibition of K+ channels. We propose that this ATP-sensitive K+ current plays an important role in the maintenance of the resting potential in arterial muscle.

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