Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes

2000 ◽  
Vol 278 (2) ◽  
pp. C352-C362 ◽  
Author(s):  
In Deok Kong ◽  
Sang Don Koh ◽  
Kenton M. Sanders
Keyword(s):  
Guinea Pig ◽  
Pyridoxal Phosphate ◽  
Receptor Blocker ◽  
Disulfonic Acid ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
Relaxation Responses ◽  
Spontaneous Transient Outward Currents

Spontaneous transient outward currents (STOCs) were recorded from smooth muscle cells of the guinea pig taenia coli using the whole cell patch-clamp technique. STOCs were resolved at potentials positive to −50 mV. Treating cells with caffeine (1 mM) caused a burst of outward currents followed by inhibition of STOCs. Replacing extracellular Ca2+ with equimolar Mn2+ caused STOCs to “run down.” Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM) inhibited large-amplitude STOCs, but small-amplitude “mini-STOCs” remained in the presence of these drugs. Mini-STOCs were reduced by apamin (500 nM), an inhibitor of small-conductance Ca2+-activated K+ channels (SK channels). Application of ATP or 2-methylthioadenosine 5′-triphosphate (2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATP persisted in the presence of charybdotoxin but were blocked by combination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did not increase STOCs in the presence of pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid, a P2 receptor blocker. Similarly, pretreatment of cells with U-73122 (1 μM), an inhibitor of phospholipase C (PLC), abolished the effects of 2-MeS-ATP. Xestospongin C, an inositol 1,4,5-trisphosphate (IP3) receptor blocker, attenuated STOCs, but these events were not affected by ryanodine. The data suggest that purinergic activation through P2Y receptors results in localized Ca2+ release via PLC- and IP3-dependent mechanisms. Release of Ca2+ is coupled to STOCs, which are composed of currents mediated by large-conductance Ca2+-activated K+ channels and SK channels. The latter are thought to mediate hyperpolarization and relaxation responses of gastrointestinal muscles to inhibitory purinergic stimulation.

scholarly journals Small-conductance Ca2+-dependent K+ channels activated by ATP in murine colonic smooth muscle

1997 ◽  
Vol 273 (6) ◽  
pp. C2010-C2021 ◽  
Author(s):  
S. D. Koh ◽  
G. M. Dick ◽  
K. M. Sanders
Keyword(s):  
Smooth Muscle ◽  
Pyridoxal Phosphate ◽  
Single Channel ◽  
Outward Current ◽  
Disulfonic Acid ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Outward Currents ◽  
Murine Colon ◽  
Small Conductance

The patch-clamp technique was used to determine the ionic conductances activated by ATP in murine colonic smooth muscle cells. Extracellular ATP, UTP, and 2-methylthioadenosine 5′-triphosphate (2-MeS-ATP) increased outward currents in cells with amphotericin B-perforated patches. ATP (0.5–1 mM) did not affect whole cell currents of cells dialyzed with solutions containing ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid. Apamin (3 × 10−7M) reduced the outward current activated by ATP by 32 ± 5%. Single channel recordings from cell-attached patches showed that ATP, UTP, and 2-MeS-ATP increased the open probability of small-conductance, Ca2+-dependent K+ channels with a slope conductance of 5.3 ± 0.02 pS. Caffeine (500 μM) enhanced the open probability of the small-conductance K+ channels, and ATP had no effect after caffeine. Pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid tetrasodium (PPADS, 10−4 M), a nonselective P2 receptor antagonist, prevented the increase in open probability caused by ATP and 2-MeS-ATP. PPADS had no effect on the response to caffeine. ATP-induced hyperpolarization in the murine colon may be mediated by P2y-induced release of Ca2+ from intracellular stores and activation of the 5.3-pS Ca2+-activated K+ channels.

Permeability of time-dependent K+ channel in guinea pig ventricular myocytes to Cs+, Na+, NH4+, and Rb+

1990 ◽  
Vol 259 (5) ◽  
pp. H1448-H1454 ◽  
Author(s):  
R. W. Hadley ◽  
J. R. Hume
Keyword(s):  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Outward Current ◽  
Time Dependent ◽  
K Channel ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Outward Currents

Currents through time-dependent K+ channels (also referred to as IK or the delayed rectifier) were studied with the whole cell patch-clamp technique in isolated guinea pig ventricular myocytes. IK measurements were restricted to the examination of deactivation tail currents. Substitution of various monovalent cations for external K+ produced shifts of the reversal potential of IK. These shifts were used to calculate permeability ratios relative to K+. The permeability sequence for the IK channels was K+ = Rb+ greater than NH4+ = Cs+ greater than Na+. Time-dependent outward currents were also examined when the myocytes were dialyzed with Cs+ instead of K+. A sizeable time-dependent outward current, quite similar to that seen with K+ dialysis, was demonstrated. This current was primarily carried by intracellular Cs+, as the reversal potential of the current shifted 46 mV per 10-fold change of external Cs+ concentration. The significance of Cs+ permeation through IK channels is discussed with respect to the common use of Cs+ in isolating other currents.

scholarly journals The effect of C-type natriuretic peptide on delayed rectifier potassium currents in gastric antral circular myocytes of the guinea-pig

2008 ◽  
pp. 55-62
Author(s):  
HY Xu ◽  
X Huang ◽  
M Yang ◽  
J-B Sun ◽  
L-H Piao ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Phosphodiesterase Inhibitor ◽  
Potassium Currents ◽  
Inhibitory Effect ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Dependent Protein ◽  
Gastric Myocytes

C-type natriuretic peptides (CNP) play an inhibitory role in smooth muscle motility of the gastrointestinal tract, but the effect of CNP on delayed rectifier potassium currents is still unclear. This study was designed to investigate the effect of CNP on delayed rectifier potassium currents and its mechanism by using conventional whole-cell patch-clamp technique in guinea-pig gastric myocytes isolated by collagenase. CNP significantly inhibited delayed rectifier potassium currents [I(K (V))] in dose-dependent manner, and CNP inhibited the peak current elicited by depolarized step pulse to 86.1+/-1.6 % (n=7, P<0.05), 78.4+/-2.6 % (n=10, P<0.01) and 67.7+/-2.3 % (n=14, P<0.01), at concentrations of 0.01 micromol/l, 0.1 micromol/l and 1 micromol/l, respectively, at +60 mV. When the cells were preincubated with 0.1 micromol/l LY83583, a guanylate cyclase inhibitor, the 1 ?micromol/l CNP-induced inhibition of I(K (V)) was significantly impaired but when the cells were preincubated with 0.1 micromol/l zaprinast, a cGMP-sensitive phosphodiesterase inhibitor, the 0.01 micromol/l CNP-induced inhibition of I(K (V)) was significantly potentiated. 8-Br-cGMP, a membrane permeable cGMP analogue mimicked inhibitory effect of CNP on I(K (V)). CNP-induced inhibition of I(K (V)) was completely blocked by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). The results suggest that CNP inhibits the delayed rectifier potassium currents via cGMP-PKG signal pathway in the gastric antral circular myocytes of the guinea-pig.

Force measurements from voltage-clamped guinea pig ventricular myocytes

1990 ◽  
Vol 258 (2) ◽  
pp. H452-H459 ◽  
Author(s):  
N. Shepherd ◽  
M. Vornanen ◽  
G. Isenberg
Keyword(s):  
Guinea Pig ◽  
Time Course ◽  
Ventricular Myocytes ◽  
Isometric Force ◽  
Contractile Force ◽  
Patch Clamp Technique ◽  
Thin Glass ◽  
Tonic Component ◽  
Time To Peak ◽  
Whole Cell Patch Clamp

We describe the first observations of isolated mammalian guinea pig ventricular myocytes that combine measurements of contractile force with the voltage-clamp method. The myocytes were attached by poly-L-lysine to the beveled ends of a pair of thin glass rods having a compliance of 0.76 m/N. The contractile force of a cell caused a 1- to 3-microm displacement of the rods; the motion of which was converted to an output voltage by phototransistors. By the use of the whole cell patch-clamp technique, the cells were depolarized at 1 Hz with 200-ms-long clamp pulses from -45 to +5 mV (35 degrees C, 3.6 mM CaCl2). Isometric force began after a latency of 7 +/- 2 ms, peaked at 93 +/- 21 ms, and relaxed (90%) at 235 +/- 63 ms. The time course of force was always faster than that of isotonic shortening (time to peak 154 +/- 18 ms). With 400-ms-long depolarizations, a tonic component was recorded as either sustained force or sustained shortening that decayed on repolarization. Substitution of Ca by Sr in the bath increased the inward current through Ca channels but slowed down the time course of force development. The results are consistent with the hypothesis that activator calcium derives mainly from internal stores and that Ca release needs Ca entry through channels.

scholarly journals ATP is involved in myocardial and vascular effects of exogenous bradykinin in ejecting guinea pig heart

1999 ◽  
Vol 277 (2) ◽  
pp. H818-H825 ◽  
Author(s):  
Peter B. Anning ◽  
Bernard D. Prendergast ◽  
Philip A. MacCarthy ◽  
Ajay M. Shah ◽  
Derek C. Buss ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Guinea Pig ◽  
Coronary Flow ◽  
Pyridoxal Phosphate ◽  
Left Ventricular ◽  
Luciferase Assay ◽  
Disulfonic Acid ◽  
Vascular Effects ◽  
Guinea Pig Heart ◽  
Nitric Oxide Release

It has recently been reported that bradykinin induces selective left ventricular (LV) relaxation in isolated guinea pig hearts via the release of nitric oxide. Exogenous bradykinin also induces vasodilation, which is only partly due to nitric oxide release. In the present study we investigated the role of adenyl purines on these bradykinin-induced effects. Isolated ejecting guinea pig hearts were studied. LV pressure was monitored by a 2-Fr micromanometer-tipped catheter. ATP concentrations were measured using a luciferin-luciferase assay. Bradykinin (1 and 100 nM) caused a progressive acceleration of LV relaxation together with a transient increase in coronary flow. These effects were inhibited by the nonselective P2 purinoceptor antagonist suramin (1 μM, n = 6) but were unaffected by the selective P2x purinoceptor antagonist pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid (1 μM, n = 6). These myocardial and vascular effects of bradykinin were associated with increased ATP levels in coronary effluent. These data suggest that the selective enhancement of LV relaxation and rise in coronary flow induced by exogenous bradykinin involve endogenous ATP and the subsequent stimulation of P2 purinoceptors.

PKC regulation of cardiac CFTR Cl− channel function in guinea pig ventricular myocytes

1998 ◽  
Vol 275 (1) ◽  
pp. C293-C302 ◽  
Author(s):  
Lisa M. Middleton ◽  
Robert D. Harvey
Keyword(s):  
Protein Kinase ◽  
Patch Clamp ◽  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Patch Clamp Technique ◽  
Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Perforated Patch Clamp

The role of protein kinase C (PKC) in regulating the protein kinase A (PKA)-activated Cl− current conducted by the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (cCFTR) was studied in guinea pig ventricular myocytes using the whole cell patch-clamp technique. Although stimulation of endogenous PKC with phorbol 12,13-dibutyrate (PDBu) alone did not activate this Cl− current, even when intracellular dialysis was limited with the perforated patch-clamp technique, activation of PKC did elicit a significant response in the presence of PKA-dependent activation of the current by the β-adrenergic receptor agonist isoproterenol. PDBu increased the magnitude of the Cl− conductance activated by a supramaximally stimulating concentration of isoproterenol by 21 ± 3.3% ( n = 9) when added after isoproterenol and by 36 ± 16% ( n= 14) when introduced before isoproterenol. 4α-Phorbol 12,13-didecanoate, a phorbol ester that does not activate PKC, did not mimic these effects. Preexposure to chelerythrine or bisindolylmaleimide, two highly selective inhibitors of PKC, significantly reduced the magnitude of the isoproterenol-activated Cl− current by 79 ± 7.7% ( n = 11) and 52 ± 10% ( n = 8), respectively. Our results suggest that although acute activation of endogenous PKC alone does not significantly regulate cCFTR Cl− channel activity in native myocytes, it does potentiate PKA-dependent responses, perhaps most dramatically demonstrated by basal PKC activity, which may play a pivotal role in modulating the function of these channels.

scholarly journals Intracellular calcium events activated by ATP in murine colonic myocytes

2000 ◽  
Vol 279 (1) ◽  
pp. C126-C135 ◽  
Author(s):  
Orline Bayguinov ◽  
Brian Hagen ◽  
Adrian D. Bonev ◽  
Mark T. Nelson ◽  
Kenton M. Sanders
Keyword(s):  
Ryanodine Receptors ◽  
Smooth Muscles ◽  
Sk Channels ◽  
Inhibitory Neurotransmitter ◽  
Outward Currents ◽  
Ionic Conductances ◽  
Visceral Muscles ◽  
Murine Colon ◽  
Spontaneous Transient Outward Currents ◽  
Purinergic Stimulation

ATP is a candidate enteric inhibitory neurotransmitter in visceral smooth muscles. ATP hyperpolarizes visceral muscles via activation of small-conductance, Ca2+-activated K+ (SK) channels. Coupling between ATP stimulation and SK channels may be mediated by localized Ca2+ release. Isolated myocytes of the murine colon produced spontaneous, localized Ca2+ release events. These events corresponded to spontaneous transient outward currents (STOCs) consisting of charybdotoxin (ChTX)-sensitive and -insensitive events. ChTX-insensitive STOCs were inhibited by apamin. Localized Ca2+ transients were not blocked by ryanodine, but these events were reduced in magnitude and frequency by xestospongin C (Xe-C), a blocker of inositol 1,4,5-trisphosphate receptors. Thus we have termed the localized Ca2+ events in colonic myocytes “Ca2+ puffs.” The P2Y receptor agonist 2-methylthio-ATP (2-MeS-ATP) increased the intensity and frequency of Ca2+ puffs. 2-MeS-ATP also increased STOCs in association with the increase in Ca2+ puffs. Pyridoxal-phospate-6-azophenyl-2′,4′-disculfonic acid tetrasodium, a P2 receptor inhibitor, blocked responses to 2-MeS-ATP. Spontaneous Ca2+ transients and the effects of 2-MeS-ATP on Ca2+ puffs and STOCs were blocked by U-73122, an inhibitor of phospholipase C. Xe-C and ryanodine also blocked responses to 2-MeS-ATP, suggesting that, in addition to release from IP3receptor-operated stores, ryanodine receptors may be recruited during agonist stimulation to amplify release of Ca2+. These data suggest that localized Ca2+ release modulates Ca2+-dependent ionic conductances in the plasma membrane. Localized Ca2+ release may contribute to the electrical responses resulting from purinergic stimulation.

Spontaneous transient outward currents and delayed rectifier K+ current: effects of hypoxia

1998 ◽  
Vol 275 (1) ◽  
pp. L145-L154 ◽  
Author(s):  
C. Vandier ◽  
M. Delpech ◽  
P. Bonnet
Keyword(s):  
Steady State ◽  
Outward Current ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
Outward Currents ◽  
K Current ◽  
The Mean ◽  
Patch Configuration ◽  
Spontaneous Transient Outward Currents ◽  
Dependent Mechanism

Single smooth muscle cells of rabbit intrapulmonary artery were voltage clamped using the perforated-patch configuration of the patch-clamp technique. We observed spontaneous transient outward currents (STOCs) and a steady-state outward current. Because STOCs were tetraethylammonium sensitive and activated by Ca2+ influx, they were believed to represent activation of Ca2+-activated K+ channels. The steady-state outward current, which was sensitive to 4-aminopyridine, was the delayed rectifier K+ current. In cells voltage clamped at 0 mV, we found that STOCs were not randomly distributed in amplitude but were composed of multiples of 1.57 ± 0.56 pA/pF. The mean frequency of STOCs was 5.51 ± 3.49 Hz. Ryanodine (10 μM), caffeine (5 mM), thapsigargin (200 nM), and hypoxia [Formula: see text] = 10 mmHg) decreased STOCs. The effect of hypoxia on STOCs was partially reversible only if the experiment was conducted in the presence of thapsigargin. Hypoxia and thapsigargin decrease steady-state outward current. Thapsigargin and removal of external Ca2+abolished the effect of hypoxia, suggesting that hypoxia decreases steady-state outward current by a Ca2+-dependent mechanism.

Calcium-activated chloride currents in primary cultures of rabbit distal convoluted tubule

1996 ◽  
Vol 271 (4) ◽  
pp. F940-F950 ◽  
Author(s):  
M. Bidet ◽  
M. Tauc ◽  
I. Rubera ◽  
G. de Renzis ◽  
C. Poujeol ◽  
...  
Keyword(s):  
Ion Selectivity ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Distal Convoluted Tubule ◽  
Disulfonic Acid ◽  
Resting Membrane Potential ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Steady State Current ◽  
Cl Permeability

Chloride (Cl-) conductances were studied in primary cultures of rabbit distal convoluted tubule (very early distal “bright” convoluted tubule, DCTb) by the whole cell patch-clamp technique. We identified a Cl- current activated by 2 microM extracellular ionomycin. The kinetics of the macroscopic current were time dependent for depolarizing potentials with a slow developing component. The steady state current presented outward rectification, and the ion selectivity sequence was I- > Br- > > Cl > glutamate. The current was inhibited by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and 1 mM diphenylamine-2-carboxylate. To identify the location of the Cl- conductance, 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence experiments were carried out in confluent cultures developed on collagen-coated permeable filters. Cl- removal from the apical solution induced a Cl- efflux that was stimulated by 10 microM forskolin. Forskolin had no effect on the basolateral Cl- permeability Cl- substitution in the basolateral solution induced an efflux stimulated by 2 microM ionomycin or 50 microM extracellular ATP Ionomycin had no effect on the apical Cl- fluxes. Thus cultured DCTb cells exhibit Ca(2+)-activated Cl- channels located in the basolateral membrane. This Cl- permeability was active at a resting membrane potential and could participate in the Cl- reabsorption across the DCTb in control conditions.

Spontaneous Ryanodine-Receptor-Dependent Ca2+-Activated K+ Currents and Hyperpolarizations in Rat Medial Preoptic Neurons

2010 ◽  
Vol 103 (5) ◽  
pp. 2900-2911 ◽  
Author(s):  
Göran Klement ◽  
Michael Druzin ◽  
David Haage ◽  
Evgenya Malinina ◽  
Peter Århem ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Ryanodine Receptors ◽  
Underlying Mechanism ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Channel Activation ◽  
Impulse Generation ◽  
Outward Currents ◽  
Main Charge ◽  
Perforated Patch Clamp

The aim of the present study was to clarify the identity of slow spontaneous currents, the underlying mechanism and possible role for impulse generation in neurons of the rat medial preoptic nucleus (MPN). Acutely dissociated neurons were studied with the perforated patch-clamp technique. Spontaneous outward currents, at a frequency of ∼0.5 Hz and with a decay time constant of ∼200 ms, were frequently detected in neurons when voltage-clamped between approximately −70 and −30 mV. The dependence on extracellular K+ concentration was consistent with K+ as the main charge carrier. We concluded that the main characteristics were similar to those of spontaneous miniature outward currents (SMOCs), previously reported mainly for muscle fibers and peripheral nerve. From the dependence on voltage and from a pharmacological analysis, we concluded that the currents were carried through small-conductance Ca2+-activated (SK) channels, of the SK3 subtype. From experiments with ryanodine, xestospongin C, and caffeine, we concluded that the spontaneous currents were triggered by Ca2+ release from intracellular stores via ryanodine receptor channels. An apparent voltage dependence was explained by masking of the spontaneous currents as a consequence of steady SK-channel activation at membrane potentials > −30 mV. Under current-clamp conditions, corresponding transient hyperpolarizations occasionally exceeded 10 mV in amplitude and reduced the frequency of spontaneous impulses. In conclusion, MPN neurons display spontaneous hyperpolarizations triggered by Ca2+ release via ryanodine receptors and SK3-channel activation. Thus such events may affect impulse firing of MPN neurons.

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