Protein kinase inhibitor H-89 reverses forskolin stimulation of cardiac L-type calcium current

1995 ◽  
Vol 268 (3) ◽  
pp. C651-C659 ◽  
Author(s):  
W. Yuan ◽  
D. M. Bers
Keyword(s):  
Protein Kinase ◽  
Patch Clamp ◽  
Kinase Inhibitor ◽  
Ventricular Myocytes ◽  
Calcium Currents ◽  
Maximal Conductance ◽  
Perforated Patch ◽  
Dependent Inactivation ◽  
Protein Kinase N ◽  
Steady State Inactivation

Calcium currents (ICa) and barium currents (IBa) were measured in freshly isolated single ferret ventricular myocytes, using the whole cell patch-clamp and perforated patch-clamp techniques with Na and K currents blocked by tetraethylammonium and Cs. The membrane potential (Em) dependence of activation and steady-state inactivation curves were determined using a Boltzmann relation, where E0.5 is the Em at half-maximal conductance. Forskolin (1 microM) increased the rate of ICa inactivation, especially in perforated patch, but slowed IBa inactivation. The acceleration is likely to be due to greater Ca-dependent inactivation of ICa, where the slowing of IBa inactivation may be due to protein kinase A-dependent slowing of Em-dependent inactivation. Forskolin (1-10 microM) also increased ICa amplitude by two- to threefold and shifted the E0.5 for both activation and inactivation to more negative potentials by 7-8 mV. The effect of forskolin on the amplitude of ICa could be reversed by an inhibitor of adenosine 3',5'-cyclic monophosphate-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; 1-10 microM). However, H-89 did not reverse the shift of E0.5 induced by forskolin. H-89 application by itself does not decrease basal ICa but does shift the E0.5 of both activation and inactivation to more negative values of Em. It is possible that H-89 reverses the shift induced by regulatory phosphorylation (due to forskolin) but induces a coincidental negative shift itself.

Modulation of ICa-L by α1-adrenergic stimulation in rat ventricular myocytes

2005 ◽  
Vol 83 (11) ◽  
pp. 1015-1024 ◽  
Author(s):  
Shetuan Zhang ◽  
Jijin Lin ◽  
Yuji Hirano ◽  
Masayasu Hiraoka
Keyword(s):  
Protein Kinase ◽  
Patch Clamp ◽  
Kinase Inhibitor ◽  
Ventricular Myocytes ◽  
Adrenergic Stimulation ◽  
Pipette Solution ◽  
Rat Ventricular Myocytes ◽  
Perforated Patch ◽  
Intracellular Ca ◽  
Perforated Patch Clamp

We found when L-type calcium current (ICa-L) was recorded with the perforated patch-clamp method in rat ventricular myocytes that bath application of phenylephrine (with propranolol) evoked a biphasic response characterized by an initial transient suppression followed by a sustained potentiation. The transient suppression occurred 30–60 s after phenylephrine perfusion and reached peak inhibition at approximately 2 min. The biphasic modulation of ICa-L was also elicited by methoxamine, and the effects of phenylephrine were blocked by prazosin, indicating that the responses were mediated through α1-adrenoceptors. Pretreatment of cells with H7 (100 µmol/L), a broad-spectrum protein kinase inhibitor that inhibits both protein kinase C and A, eliminated potentiation but did not affect transient suppression. The transient suppression occurred concurrently with the acceleration of the fast component of ICa-L inactivation. Depletion of intracellular Ca2+ stores by ryanodine plus caffeine or thapsigargin eliminated the transient suppression. When ICa-L was recorded with whole-cell patch-clamp and with 0.05 mmol/L EGTA in the pipette solution to allow intracellular Ca2+ to fluctuate, phenylephrine evoked a transient suppression as in the perforated patch recordings. Heparin, a specific blocker of IP3 (inositol 1,4,5-trisphosphate) receptors, eliminated the phenylephrine-induced transient suppression of ICa-L when added to the pipette solution. Intensive chelation of intracellular Ca2+ by 5 mmol/L BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) in the pipette solution also eliminated the phenylephrine-induced transient suppression of ICa-L. We conclude that transient increase in the concentration of intracellular calcium ([Ca2+]i) caused by Ca2+ release from intracellular stores underlies the transient suppression of ICa-L, whereas the potentiation of ICa-L is a result of activation of protein kinases.Key words: Ca2+ mobilization, IP3, Ca2+-induced inactivation of Ca2+ current, perforated patch-clamp.

Intracellular Cs+ activates the PKA pathway, revealing a fast, reversible, Ca2+-dependent inactivation of L-type Ca2+ current

2003 ◽  
Vol 285 (2) ◽  
pp. C310-C318 ◽  
Author(s):  
Fabien Brette ◽  
Alain Lacampagne ◽  
Laurent Sallé ◽  
Ian Findlay ◽  
Jean-Yves Le Guennec
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Ventricular Myocytes ◽  
Cyclopiazonic Acid ◽  
Dependent Protein Kinase ◽  
Pipette Solution ◽  
Camp Dependent Protein Kinase ◽  
Dependent Inactivation ◽  
Dependent Protein ◽  
Pka Pathway

Inactivation of the L-type Ca2+ current ( ICaL) was studied in isolated guinea pig ventricular myocytes with different ionic solutions. Under basal conditions, ICaL of 82% of cells infused with Cs+-based intracellular solutions showed enhanced amplitude with multiphasic decay and diastolic depolarization-induced facilitation. The characteristics of ICaL in this population of cells were not due to contamination by other currents or an artifact. These phenomena were reduced by ryanodine, caffeine, cyclopiazonic acid, the protein kinase A inhibitor H-89, and the cAMP-dependent protein kinase inhibitor. Forskolin and isoproterenol increased ICaL by only ∼60% in these cells. Cells infused with either N-methyl-d-glucamine or K+-based intracellular solutions did not show multiphasic decay or facilitation under basal conditions. Isoproterenol increased ICaL by ∼200% in these cells. In conclusion, we show that multiphasic inactivation of ICaL is due to Ca2+-dependent inactivation that is reversible on a time scale of tens of milliseconds. Cs+ seems to activate the cAMP-dependent protein kinase pathway when used as a substitute for K+ in the pipette solution.

Nitric oxide and cGMP protein kinase activity in aged ventricular myocytes

2001 ◽  
Vol 281 (6) ◽  
pp. H2304-H2309 ◽  
Author(s):  
Qihang Zhang ◽  
Bruno Molino ◽  
Lin Yan ◽  
Todd Haim ◽  
Yakir Vaks ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Ventricular Myocytes ◽  
Nitric Oxide Donor ◽  
Protein Kinase Activity ◽  
Edge Detector ◽  
Negative Effects ◽  
Dependent Protein

We tested the hypothesis that nitric oxide-induced negative functional effects through cGMP would be reduced in aged cardiac myocytes. Maximum rate of shortening ( R max) and percent shortening of ventricular myocytes from young (6 mo) and old (3 y) rabbits were studied using a video edge detector. cGMP-dependent phosphorylation was examined by electrophoresis and autoradiography. Myocytes received a nitric oxide donor S-nitroso- N-acetyl-penicillamine (SNAP, 10−7, 10−6, and 10−5 M) followed by KT-5823 (10−6 M), a cGMP protein kinase inhibitor. Baseline function was similar in young and old myocytes (89.1 ± 4.5 young vs. 86.4 ± 8.3 μm/s old R max, 5.6 ± 0.3 vs. 5.2 ± 0.7%shortening). SNAP (10−5 M) decreased R max in both young (25%, n = 6) and old myocytes (24%, n = 7). SNAP also reduced percent shortening by 28% in young and 23% in old myocytes. The negative effects of SNAP were partially reversed by KT-5823 only in young myocytes. Multiple proteins were phosphorylated by cGMP, and KT-5823 could reduce this effect. The degree of phosphorylation was significantly less in old myocytes. These results suggest that the functional response of ventricular myocytes to nitric oxide was preserved during aging. However, the importance of cGMP-dependent protein phosphorylation was decreased, indicating a shift to other pathways.

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.

Altered beta-adrenergic and muscarinic response of CFTR Cl- current in dialyzed cardiac myocytes

1995 ◽  
Vol 268 (5) ◽  
pp. H1795-H1802
Author(s):  
S. I. Zakharov ◽  
R. D. Harvey
Keyword(s):  
Patch Clamp ◽  
Muscarinic Receptor ◽  
Ventricular Myocytes ◽  
Receptor Activation ◽  
Current Response ◽  
Patch Clamp Technique ◽  
Beta Adrenergic ◽  
Perforated Patch ◽  
Clamp Technique ◽  
Perforated Patch Clamp

Autonomic regulation of the cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current was studied in isolated guinea pig ventricular myocytes using various configurations of the whole cell patch-clamp technique. When currents were recorded using the conventional patch-clamp technique, it was possible to continue to activate the Cl- current on repeated exposure to isoproterenol (Iso) for up to 60 min after initiating dialysis. However, there was significant rundown of the magnitude of the Cl- current response to the maximally stimulating concentrations of Iso. In addition, the concentration of Iso that produced half-maximal activation of the Cl- current (K1/2) increased with time. Conversely, the K1/2 for acetylcholine inhibition of the Iso-activated current decreased with time. When currents were recorded using the perforated patch-clamp technique, the sensitivity to both beta-adrenergic- and muscarinic-receptor stimulation was stable. Immediately after initiation of dialysis with the conventional patch-clamp technique, the sensitivity to Iso was nearly identical to that determined using the perforated patch-clamp technique. However, the initial sensitivity to muscarinic-receptor activation was significantly greater. These results indicate that cell dialysis associated with conventional patch-clamp techniques not only results in a time-dependent rundown of current amplitude, but it also significantly alters the concentration dependence of beta-adrenergic and muscarinic-receptor regulation of ion channel function.

Role of Ca2+/calmodulin-dependent protein kinase II in the regulation of the cardiac L-type Ca2+ current during endothelin-1 stimulation

2010 ◽  
Vol 298 (6) ◽  
pp. H1902-H1907 ◽  
Author(s):  
Kimiaki Komukai ◽  
Jin O-Uchi ◽  
Satoshi Morimoto ◽  
Makoto Kawai ◽  
Kenichi Hongo ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Patch Clamp ◽  
Receptor Antagonist ◽  
Ventricular Myocytes ◽  
Receptor Protein ◽  
Dependent Protein Kinase ◽  
Rat Ventricular Myocytes ◽  
Endothelin 1 ◽  
Protein Kinase Ii ◽  
Dependent Protein

Endothelin-1 (ET-1) shows a positive inotropic effect on cardiac muscle. Although the L-type Ca2+ current ( ICa) is one of the important determinants of cardiac excitation-contraction coupling, the effect of ET-1 on the ICa is not always clear. The controversial results appear to be due to different patch-clamp methods. The present study measured the effect of ET-1 on the ICa of rat ventricular myocytes using the perforated patch-clamp technique. The holding potential was set to −40 mV, and depolarization was applied every 10 s. ET-1 (10 nM) increased the ICa in a monophasic manner. The current reached a steady state 15 min after the application of ET-1, when the measurement was done. Endothelin receptor subtype expression was also investigated using Western immunoblotting. ETA-receptor protein was expressed, but ETB-receptor protein was not expressed, in the cell membranes of rat ventricular myocytes. The effect of ET-1 on the ICa was inhibited by a selective ETA-receptor antagonist, BQ-123, but not by a selective ETB-receptor antagonist, BQ-788. The effect was inhibited by protein kinase C (PKC) inhibitor chelerythrine and Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93, but not by its inactive analog KN-92. The effect of ET-1 was also blocked by another CaMKII inhibitor, autocamtide-2-related inhibitory peptide. These results suggest that ET-1 increases the ICa via the ETA-receptor-PKC-CaMKII pathway.

Stimulatory effect of ouabain on T- and L-type calcium currents in guinea pig cardiac myocytes

1990 ◽  
Vol 258 (5) ◽  
pp. H1620-H1623 ◽  
Author(s):  
B. Le Grand ◽  
E. Deroubaix ◽  
A. Coulombe ◽  
E. Coraboeuf
Keyword(s):  
Steady State ◽  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Calcium Currents ◽  
High Threshold ◽  
Inactivation Curve ◽  
Cell Recording ◽  
Steady State Inactivation ◽  
Low Threshold ◽  
Free Media

The effect of 10(-6) M ouabain on macroscopic low-threshold T-type Ca2+ and high-threshold L-type Ca2+ currents was studied by whole cell recording in isolated guinea pig ventricular myocytes superfused with K-free, Na-free media, i.e., after suppression of Na-K-ATPase activity and Na influx through the Na-Ca exchanger. Under such conditions, the amplitudes of the two currents were significantly increased by ouabain. In particular, the current occurring in the -50 to -20 mV range (T-type Ca2+) was increased two- to threefold by ouabain and suppressed by 40 microM Ni2+. Ouabain shifted by approximately 10 mV toward negative potentials the steady-state inactivation curve of the T-type Ca2+ current but not that of the L-type Ca2+ current. It is concluded that ouabain enhances not only L-type Ca2+ current but also T-type Ca2+ current possibly through different mechanisms.

Abstract 1514: RXP-E: A Connexin43-Binding Peptide that Prevents Action Potential Propagation Block

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Rebecca Lewandowski ◽  
Kristina Procida ◽  
Ravi Vaidyanathan ◽  
José Jalife ◽  
Morten S Nielsen ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Action Potential ◽  
Rhode Island ◽  
Ventricular Myocytes ◽  
Heart Association ◽  
Calcium Currents ◽  
Neonatal Rat ◽  
List Type ◽  
Resistance Pathway

Gap junctions (GJ’s) provide a low resistance pathway for cardiac electrical propagation. The role of GJ regulation in arrhythmias is unclear, partly due to the limited availability of pharmacological tools. Recently, we showed that a peptide called “RXP-E” binds to the carboxyl terminal of connexin43 (Cx43) and prevents chemically-induced uncoupling in Cx43-expressing N2a cells. Here, we used pull-down experiments to show that RXP-E binds to adult cardiac Cx43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes (NRVM’s). Separately, RXP-E was concatenated to a cytoplasmic transduction peptide for translocation into the cytoplasm (construct CTP-RXP-E). The effect of RXP-E on action potential (AP) propagation was assessed by high resolution optical mapping in monolayers of NRVMs, containing approximately 20% of randomly distributed myofibroblasts. Conduction velocity (CV) was 164 ± 8mm/sec (avg±SEM; n=12; pacing frequency 2Hz) in untreated cells, and 158±10mm/sec, (n=6) and 180±7mm/sec (n=10) in monolayers treated with CTP-RXPE or a scrambled version of the peptide (CTP-Scr), respectively (pNS). Exposure of either untreated, or CTP-Scr-treated monolayers to heptanol caused propagation block. However, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP-RXP-E, AP propagation was maintained, albeit at a slower velocity (87±5mm/sec;n=4; P<0.001). Similarly, intracellular acidification (pHi=6.2) caused a loss of AP propagation in control or CTP-Scr monolayers; however, propagation was maintained in CTP-RXP-E treated cells, though at a slower rate (CV=93 ± 28mm/sec; n=4). Consistent with these results, patch clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium or calcium currents, nor did it alter the voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to: bind cardiac Cx43; prevent heptanol and acidification-induced uncoupling of cardiac GJ’s and preserve AP propagation among cardiac myocytes. RXP-E can be used to characterize the role of GJ’s in the function of multicellular systems, including the heart. This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).

Selective disruption by protein kinases of G-protein-mediated Ca2+ channel modulation

1996 ◽  
Vol 76 (1) ◽  
pp. 311-320 ◽  
Author(s):  
M. S. Shapiro ◽  
J. Zhou ◽  
B. Hille
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Kinase Inhibitor ◽  
Sympathetic Neurons ◽  
Calcium Currents ◽  
Muscarinic Agonists ◽  
Whole Cell Patch Clamp ◽  
Cervical Ganglion ◽  
K Current ◽  
M1 Muscarinic

1. We studied the effects of phorbol-12-myristate, 13-acetate (PMA) on G-protein-mediated inhibition of Ca2+ channels by several neurotransmitters in rat superior cervical ganglion (SCG) sympathetic neurons, with the use of the whole cell patch clamp. PMA attenuated membrane-delimited inhibition of calcium currents (ICa) by norepinephrine (NE) and somatostatin by more than half, but did not attenuate inhibition by M1 muscarinic receptors, which use a diffusible cytoplasmic messenger. Inhibition of ICa by NE through pertussis-toxin-sensitive and -insensitive G proteins was equally attenuated by PMA. PMA enhanced ICa in about half the neurons (enhancement of 10 +/- 1%, mean +/- SE) and strongly reduced the holding current in 44 of 61 cells. 2. The M-type K+ current (IM) was not suppressed by PMA, and PMA did not attenuate inhibition of IM by muscarinic agonists, which is also via a diffusible cytoplasmic messenger. 3. Attenuation of NE and somatostatin inhibition by PMA was blocked by 1 microM staurosporine, a broad-spectrum protein kinase inhibitor. Tests with three inhibitors selective for distinct isoforms of protein kinase C (PKC) gave mixed results. PMA's actions were unaffected by 1 microM calphostin C, blocked by 500 nM bisindolylmaleimide, and unaffected by the pseudosubstrate inhibitor PKC19-36. 4. Thus we find that two membrane-delimited signaling pathways that inhibit ion channels in rat SCG neurons are strongly attenuated by PMA, but signaling pathway(s) that use a diffusible cytoplasmic messenger are not. We speculate that a nonstandard PKC isoform, perhaps PKC mu, mediates PMA actions.

T-type calcium currents in rat cardiomyocytes during postnatal development: contribution to hormone secretion

2000 ◽  
Vol 279 (5) ◽  
pp. H2540-H2548 ◽  
Author(s):  
Valérie Leuranguer ◽  
Arnaud Monteil ◽  
Emmanuel Bourinet ◽  
Govindan Dayanithi ◽  
Joël Nargeot
Keyword(s):  
Functional Properties ◽  
Ventricular Myocytes ◽  
Hormone Secretion ◽  
Calcium Currents ◽  
Inactivation Kinetics ◽  
Peak Current Density ◽  
Rat Cardiomyocytes ◽  
Genes Encoding ◽  
Steady State Inactivation ◽  
Postnatal Evolution

T-type Ca2+ channels have been suggested to play a role in cardiac automaticity, cell growth, and cardiovascular remodeling. Although three genes encoding for a T-type Ca2+ channel have been identified, the nature of the isoform(s) supporting the cardiac T-type Ca2+ current ( I Ca,T) has not yet been determined. We describe the postnatal evolution of I Ca,T density in freshly dissociated rat atrial and ventricular myocytes and its functional properties at peak current density in young atrial myocytes. I Ca,T displays a classical low activation threshold, rapid inactivation kinetics, negative steady-state inactivation, slow deactivation, and the presence of a window current. Interestingly, I Ca,T is poorly sensitive to Ni2+ and insensitive to R-type current toxin SNX-482. RT-PCR experiments and comparison of functional properties with recombinant Ca2+ channel subtypes suggest that neonatal I Ca,T is related to the α1G-subunit. Atrial natriuretic factor (ANF) secretion was measured using peptide radioimmunoassays in atrial tissue. Pharmacological dissection of ANF secretion indicates an important contribution of I Ca,T to Ca2+signaling during the neonatal period.

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