cGMP-mediated inhibition of cardiac L-type Ca2+current by a monoclonal antibody against the M2 ACh receptor

2001 ◽  
Vol 281 (4) ◽  
pp. C1251-C1258 ◽  
Author(s):  
J. H. M. Nascimento ◽  
L. Sallé ◽  
J. Hoebeke ◽  
J. Argibay ◽  
N. Peineau
Keyword(s):  
Monoclonal Antibody ◽  
Ventricular Myocytes ◽  
Phosphodiesterase Inhibitor ◽  
Inhibitory Effect ◽  
Intracellular Camp ◽  
Classical Pathway ◽  
Patch Clamp Technique ◽  
Cyclic Monophosphate ◽  
Whole Cell Patch Clamp ◽  
Dependent Protein

The effects of a monoclonal antibody (B8E5) directed against the second extracellular loop of the muscarinic M2receptor were studied on the L-type Ca2+ currents ( I Ca,L) of guinea pig ventricular myocytes using the whole cell patch-clamp technique. Similar to carbachol, B8E5 reduced the isoproterenol (ISO)-stimulated I Ca,L but did not significantly affect basal I Ca,L. Atropine blocked the inhibitory effect of B8E5. The electrophysiological parameters of ISO-stimulated I Ca,L were not modified in presence of B8E5. Inhibition of I Ca,L by B8E5 was still observed when intracellular cAMP was either enhanced by forskolin or maintained constant by using a hydrolysis-resistant cAMP analog (8-bromoadenosine 3′,5′-cyclic monophosphate) or by applying the phosphodiesterase inhibitor IBMX. The effect of B8E5 was mimicked by 8-bromoguanosine 3′,5′-cyclic monophosphate, a potent stimulator of cGMP-dependent protein kinase, and prevented by a selective inhibitor of nitric oxide-sensitive guanylyl cyclase {1 H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one}. These results indicate that the antibody B8E5 inhibits the β-adrenergic-stimulated I Ca,L through activation of the M2 muscarinic receptor and further suggest that the antibody acts not via the classical pathway of decreasing intracellular cAMP, but rather by increasing cGMP.

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.

scholarly journals Inhibitory Effect of Cinobufagin on L-Type Ca2+Currents, Contractility, and Ca2+Homeostasis of Isolated Adult Rat Ventricular Myocytes

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Pinya Li ◽  
Qiongtao Song ◽  
Tao Liu ◽  
Zhonglin Wu ◽  
Xi Chu ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Cell Contractility ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Time To Peak ◽  
Whole Cell Patch Clamp

Cinobufagin (CBG), a major bioactive ingredient of the bufanolide steroid compounds of Chan Su, has been widely used to treat coronary heart disease. At present, the effect of CBG on the L-type Ca2+current (ICa-L) of ventricular myocytes remains undefined. The aim of the present study was to characterize the effect of CBG on intracellular Ca2+([Ca2+]i) handling and cell contractility in rat ventricular myocytes. CBG was investigated by determining its influence onICa-L, Ca2+transient, and contractility in rat ventricular myocytes using the whole-cell patch-clamp technique and video-based edge-detection and dual-excitation fluorescence photomultiplier systems. The dose of CBG (10−8 M) decreased the maximal inhibition of CBG by 47.93%. CBG reducedICa-Lin a concentration-dependent manner with an IC50of 4 × 10−10 M, upshifted the current-voltage curve ofICa-L, and shifted the activation and inactivation curves ofICa-Lleftward. Moreover, CBG diminished the amplitude of the cell shortening and Ca2+transients with a decrease in the time to peak (Tp) and the time to 50% of the baseline (Tr). CBG inhibited L-type Ca2+channels, and reduced[Ca2+]iand contractility in adult rat ventricular myocytes. These findings contribute to the understanding of the cardioprotective efficacy of CBG.

Effects of PDE inhibitors and carbachol on the L-type Ca current in guinea pig ventricular myocytes

1993 ◽  
Vol 265 (4) ◽  
pp. H1353-H1363 ◽  
Author(s):  
K. Mubagwa ◽  
T. Shirayama ◽  
M. Moreau ◽  
A. J. Pappano
Keyword(s):  
Guinea Pig ◽  
Time Course ◽  
Ventricular Myocytes ◽  
Phosphodiesterase Inhibitor ◽  
Phosphodiesterase Inhibitors ◽  
Cell Voltage ◽  
Ca Current ◽  
Inactivation Curve ◽  
Cyclic Monophosphate ◽  
Dependent Protein

The phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX; 100 microM) and papaverine (100 microM) increased peak L-type Ca current (ICa) more than fivefold in a way similar to isoproterenol, forskolin, or intracellular adenosine 3',5'-cyclic monophosphate in guinea pig ventricular myocytes studied with the whole cell voltage-clamp technique at 22-24 degrees C. IBMX and papaverine could also induce a chloride current. Both drugs caused an apparent increase of ICa inactivation as revealed by 1) a negative shift of the ICa inactivation curve between -40 and 0 mV and 2) a suppression of the relief from inactivation at potentials positive to 0 mV. In the presence of IBMX or papaverine, the amplitudes of both the rapidly and slowly inactivating components of ICa were increased; the effect on the fast component was more pronounced. The drugs did not accelerate the inactivation time course of either component. Carbachol (CCh; 100 microM) reversed the increase in ICa produced by IBMX or papaverine. However, ICa could not be restored to its original magnitude on washout of CCh in the presence of phosphodiesterase inhibitors. In pertussis toxin-treated cells or in the presence of Ly-83583 (1-100 microM), IBMX retained its effect but CCh was unable to reduce ICa. Dialysis with guanosine 3',5'-cyclic monophosphate (cGMP; 0.1-100 microM) or 8-bromoguanosine 3',5'-cyclic monophosphate (30 microM) suppressed the increase of ICa by IBMX; the inhibition by cGMP was additive with that produced by CCh. We suggest that the major part of IBMX and papaverine effect is mediated by phosphodiesterase inhibition and involves an increase in intracellular adenosine 3',5'-cyclic monophosphate levels. CCh reversal of phosphodiesterase inhibitor action probably involves an elevation of cGMP levels and activation of cGMP-dependent protein kinase.

Fluid pressure modulates L-type Ca2+ channel via enhancement of Ca2+-induced Ca2+ release in rat ventricular myocytes

2008 ◽  
Vol 294 (4) ◽  
pp. C966-C976 ◽  
Author(s):  
Sunwoo Lee ◽  
Joon-Chul Kim ◽  
Yuhua Li ◽  
Min-Jeong Son ◽  
Sun-Hee Woo
Keyword(s):  
Ventricular Myocytes ◽  
Fluid Pressure ◽  
Inhibitory Effect ◽  
Cellular Mechanism ◽  
Confocal Imaging ◽  
Rat Ventricular Myocytes ◽  
Whole Cell Patch Clamp ◽  
Current Voltage ◽  
Intracellular Ca ◽  
High Concentrations

This study examines whether fluid pressure (FP) modulates the L-type Ca2+ channel in cardiomyocytes and investigates the underlying cellular mechanism(s) involved. A flow of pressurized (∼16 dyn/cm2) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes using a microperfusion method. The Ca2+ current ( ICa) and cytosolic Ca2+ signals were measured using a whole cell patch-clamp and confocal imaging, respectively. It was found that the FP reversibly suppressed ICa (by 25%) without altering the current-voltage relationships, and it accelerated the inactivation of ICa. The level of ICa suppression by FP depended on the level and duration of pressure. The Ba2+ current through the Ca2+ channel was only slightly decreased by the FP (5%), suggesting an indirect inhibition of the Ca2+ channel during FP stimulation. The cytosolic Ca2+ transients and the basal Ca2+ in field-stimulated ventricular myocytes were significantly increased by the FP. The effects of the FP on the ICa and on the Ca2+ transient were resistant to the stretch-activated channel inhibitors, GsMTx-4 and streptomycin. Dialysis of myocytes with high concentrations of BAPTA, the Ca2+ buffer, eliminated the FP-induced acceleration of ICa inactivation and reduced the inhibitory effect of the FP on ICa by ≈80%. Ryanodine and thapsigargin, abolishing sarcoplasmic reticulum Ca2+ release, eliminated the accelerating effect of FP on the ICa inactivation, and they reduced the inhibitory effect of FP on the ICa. These results suggest that the fluid pressure indirectly suppresses the Ca2+ channel by enhancing the Ca2+-induced intracellular Ca2+ release in rat ventricular myocytes.

Abstract 17193: Inhibiting Late Sodium Current Attenuates Isoproterenol-Induced Transient Inward Current and Delayed Afterdepolarizations in Ventricular Myocytes

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Yejia Song ◽  
Nesrine El-Bizri ◽  
Sridharan Rajamani ◽  
Luiz Belardinelli
Keyword(s):  
Ventricular Myocytes ◽  
Sodium Current ◽  
Selective Inhibition ◽  
Adrenergic Stimulation ◽  
Patch Clamp Technique ◽  
Cardiac Tissues ◽  
Inward Current ◽  
Transient Inward Current ◽  
Whole Cell Patch Clamp ◽  
Series Of Experiments

Introduction: The β-adrenergic agonist isoproterenol (ISO) is known to induce the arrhythmogenic transient inward current (I Ti ) and delayed afterdepolarization (DAD) via a stimulation of L-type Ca 2+ current. Recent studies found that ISO-induced DADs in cardiac tissues are inhibited by GS967, a selective blocker of the late Na + current (I NaL ). Thus, we hypothesize that I NaL contributes to the actions of ISO, and selective inhibition of this current will reduce ISO-induced I Ti and DADs. Methods: Transmembrane currents and action potentials of rabbit and guinea pig (GP) ventricular myocytes were recorded using the whole-cell patch-clamp technique. ISO (0.1 μM), GS967 (1 μM) and the Na + channel blocker tetrodotoxin (TTX, 3 μM) were used in the experiments. Results: In rabbit myocytes, application of ISO caused an increase in the amplitude of I NaL from -0.10±0.03 to -0.32±0.04 pA/pF (n = 17, p < 0.05). The ISO-stimulated I NaL was inhibited by GS967 and TTX. In one series of experiments, ISO increased the I NaL from -0.14±0.04 to -0.35±0.06 pA/pF, and GS967 applied in the presence of ISO reduced the current to -0.14±0.03 pA/pF (n = 9, p < 0.05). In another series of experiments, the amplitude of I NaL was increased by ISO from -0.17±0.08 to -0.41±0.09 pA/pF, and was decreased to -0.09±0.08 pA/pF when TTX was applied with ISO (n = 5, p < 0.05). Application of ISO also induced I Ti and DADs. GS967 applied in the presence of ISO inhibited the amplitude of I Ti by 52±6%, from -1.79±0.30 to -0.87±0.16 pA/pF (n = 8, p < 0.05). Consistent with the inhibition of I Ti , GS967 suppressed the amplitude of ISO-induced DADs by 56±12%, from 6.54±1.59 to 3.22±1.27 mV (n = 5, p < 0.05). Similarly, in GP myocytes ISO-induced I Ti and DADs were decreased by GS967 from -1.14±0.21 to -0.73±0.16 pA/pF (n = 7, p < 0.05) and from 7.16±0.59 to 4.67±0.24 mV (n = 5, p < 0.05), respectively. Conclusions: An increased I NaL is likely to contribute to the proarrhythmic effects of ISO in cardiac myocytes. GS967 significantly attenuated ISO-induced I NaL , I Ti and DADs, suggesting that inhibiting this current could be an effective strategy to antagonize the arrhythmogenic actions of β-adrenergic stimulation.

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.

VIP inhibits basal and histamine-stimulated proliferation of human airway smooth muscle cells

1995 ◽  
Vol 268 (6) ◽  
pp. L1047-L1051 ◽  
Author(s):  
K. Maruno ◽  
A. Absood ◽  
S. I. Said
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Thymidine Incorporation ◽  
Inhibitory Effect ◽  
Airway Smooth Muscle Cells ◽  
Dependent Manner ◽  
Cyclic Monophosphate ◽  
Cell Counts ◽  
Dependent Protein ◽  
Camp Levels

Airway smooth muscle (ASM) cell proliferation contributes to increased airway resistance in bronchial asthma. We have examined the modulation of ASM proliferation by vasoactive intestinal peptide (VIP), a cotransmitter of airway relaxation. Human ASM cells were grown in culture as a monolayer. VIP (1.0 nM-1.0 microM) inhibited proliferation in a dose-dependent manner by up to 82% on day 2, but the related peptide glucagon had no effect. Histamine (100 nM-100 microM) increased cell counts by 66%, but in the presence of VIP, cell counts and [3H]thymidine incorporation were reduced by up to 55%. Adenosine 3',5'-cyclic monophosphate (cAMP)-promoting agents, including 3-isobutyl-1-methylxanthine, forskolin, and 8-bromo-adenosine 3',5'-cyclic monophosphate, alone and especially combined with VIP, reduced cell counts and [3H]thymidine incorporation, in correlation with cAMP levels. KT-5720 (1.0 nM-1.0 microM), a selective inhibitor of cAMP-dependent protein kinase A (PKA), abolished the inhibitory effect of VIP. The results show that VIP selectively and potently inhibits human ASM cell growth and multiplication, and nullifies the mitogenic effect of histamine, by a PKA-mediated mechanism. A deficiency of VIP may lead to ASM hyperplasia due to unopposed stimulation by endogenous mitogens.

Properties of voltage-gated Ca2+ channels in rabbit ventricular myocytes expressing Ca2+ channel α1E cDNA

2001 ◽  
Vol 280 (1) ◽  
pp. C175-C182 ◽  
Author(s):  
Michihiro Tateyama ◽  
Shuqin Zong ◽  
Tsutomu Tanabe ◽  
Rikuo Ochi
Keyword(s):  
Cardiac Myocytes ◽  
Fluorescent Protein ◽  
Ventricular Myocytes ◽  
Foreign Gene ◽  
Adrenergic Stimulation ◽  
Patch Clamp Technique ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Green Fluorescent ◽  
Ca2 Channels

Using the whole-cell patch-clamp technique, we have studied the properties of α1ECa2+ channel transfected in cardiac myocytes. We have also investigated the effect of foreign gene expression on the intrinsic L-type current ( I Ca,L). Expression of green fluorescent protein significantly decreased the I Ca,L. By contrast, expression of α1E with β2b and α2/δ significantly increased the total Ca2+ current, and in these cells a Ca2+ antagonist, PN-200-110 (PN), only partially blocked the current. The remaining PN-resistant current was abolished by the application of a low concentration of Ni2+and was little affected by changing the charge carrier from Ca2+ to Ba2+ or by β-adrenergic stimulation. On the basis of its voltage range for activation, this channel was classified as a high-voltage activated channel. Thus the expression of α1E did not generate T-like current in cardiac myocytes. On the other hand, expression of α1E decreased I Ca,L and slowed the I Ca,L inactivation. This inactivation slowing was attenuated by the β2b coexpression, suggesting that the α1E may slow the inactivation of I Ca,L by scrambling with α1C for intrinsic auxiliary β.

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.

Effects of cAMP on serotonin evoked calcium transients in cultured rat airway smooth muscle cells

1997 ◽  
Vol 272 (5) ◽  
pp. L865-L871 ◽  
Author(s):  
B. Tolloczko ◽  
Y. L. Jia ◽  
J. G. Martin
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Airway Smooth Muscle ◽  
Airway Smooth Muscle Cells ◽  
Intracellular Camp ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Dependent Protein ◽  
High Concentrations

Agents increasing intracellular adenosine 3',5'-cyclic monophosphate (cAMP) cause relaxation of airway smooth muscle. However, the mechanisms of their action are not fully understood. We investigated the role of cAMP in the modulation of intracellular Ca2+ concentration ([Ca2+]i) transients evoked by serotonin (5-HT) in cultured rat tracheal smooth muscle (TSM) cells. Forskolin (10(-7) M) caused a significant elevation of intracellular cAMP and a 60% relaxation of tracheal rings contracted with 5-HT but did not affect [Ca2+]i in TSM cells. Forskolin (10(-5) M) completely relaxed tracheal rings and significantly decreased [Ca2+]i during the sustained phase of the 5-HT response. Forskolin-induced relaxation was attenuated by the cAMP-dependent protein kinase A (PKA) inhibitor Rp diastereomer of cAMP (Rp-cAMPS; 10(-4) M) and by the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor [Rp isomer of 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate, 10(-4) M]. The effects of forskolin on [Ca2+]i were not altered by the PKA inhibitor but were abolished by the PKG inhibitor and thapsigargin. These results indicate that, in rat TSM, the relaxant effects of high concentrations of cAMP may be mediated, at least in part, by facilitating the sequestration of Ca2+ into intracellular stores by a mechanism involving PKG.

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