Modulation of L-type Ca2+ channels in neonatal rat heart by a novel Ca2+ channel agonist

2003 ◽  
Vol 81 (2) ◽  
pp. 135-141
Author(s):  
Mohamed Chahine ◽  
Adrian Sculptoreanu ◽  
Daya R Varma
Keyword(s):  
Threshold Concentration ◽  
Neonatal Rat ◽  
Maximum Effect ◽  
Heart Cells ◽  
Maximal Effect ◽  
Patch Clamp Technique ◽  
Cardiotonic Agent ◽  
Intracellular Ca ◽  
Current Activation ◽  
Cgp 48506

L-type Ca2+ channels are essential in triggering the intracellular Ca2+ release and contraction in heart cells. In this study, we used patch clamp technique to compare the effect of two pure enantiomers of L-type Ca2+ channel agonists: (+)-CGP 48506 and the dihydropyridine (+)-SDZ-202 791 in cardiomyocytes from rats 2–5 days old. The predominant Ca2+ current activated by standard step pulses in these myocytes was L-type Ca2+ current. The di hy dro py ri dine antagonist (+)-PN200-110 (5 μM) blocked over 90% of Ca2+ currents in most cells tested. CGP 48506 lead to a maximum of 200% increase in currents. The threshold concentration for the CGP effect was at 1 μM and the maximum was reached at 20 μM. SDZ-202 791 had effects in nanomolar concentrations and a maximum effect at about 2 μM. The maximal effect of (+)-SDZ-202 791 was a 400% increase in the amplitude of Ca2+ currents and was accompanied by a 10–15 mV leftward shift in the voltage dependence of activation. CGP 48506 increased the currents equally at all voltages tested. Both compounds slowed the deactivation of tail currents and lead to the appearance of slowly activating and slowly deactivating current components. However, SDZ-202 791 had larger effects on deactivation and CGP 48506 had larger effect on the rate of Ca2+ current activation. The effect of SDZ-202 791 was fully additive to that of CGP 48506 even after maximum concentrations of CGP. This observation suggests that the two Ca2+ channel agonists may act at two different sites on the L-type Ca2+ channel. We suggest that CGP 48506 would be a potential cardiotonic agent without the deleterious proarrhythmic effects attributable to the dihydropyridine agonists.Key words: heart failure, calcium channels, dihydropyridine, CGP 48506 (5-methyl-6-phenyl-1, 3,5,6-tetra hydro-3,6-methano-1,5-benzodiazocine-2,4-dione).

scholarly journals Mobilization of intracellular calcium by intracellular flash photolysis of caged dihydrosphingosine in cultured neonatal rat sensory neurones.

1998 ◽  
Vol 45 (2) ◽  
pp. 311-326 ◽  
Author(s):  
A Ayar ◽  
N M Thatcher ◽  
U Zehavi ◽  
D R Trentham ◽  
R H Scott
Keyword(s):  
Flash Photolysis ◽  
Reversal Potential ◽  
Neonatal Rat ◽  
Xenon Lamp ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Sensory Neurones ◽  
Whole Cell ◽  
Inward Currents ◽  
Current Activation

The ability of dihydrosphingosine to release Ca2+ from intracellular stores in neurones was investigated by combining the whole cell patch clamp technique with intracellular flash photolysis of caged, N-(2-nitrobenzyl)dihydrosphingosine. The caged dihydrosphingosine (100 microM) was applied to the intracellular environment via the CsCl-based patch pipette solution which also contained 0.3% dimethylformamide and 2 mM dithiothreitol. Cultured dorsal root ganglion neurones from neonatal rats were voltage clamped at -90 mV and inward whole cell Ca2+-activated currents were recorded in response to intracellular photorelease of dihydrosphingosine. Intracellular photorelease of dihydrosphingosine (about 5 microM) was achieved using a Xenon flash lamp. Inward Ca2+-activated currents were evoked in 50 out of 57 neurones, the mean delay to current activation following photolysis was 82+/-13 s. The responses were variable with neurones showing transient, oscillating or sustained inward currents. High voltage-activated Ca2+ currents evoked by 100 ms voltage step commands to 0 mV were not attenuated by photorelease of dihydrosphingosine. Controls showed that alone a flash from the Xenon lamp did not activate currents, and that the unphotolysed caged dihydrosphingosine, and intracellular photolysis of 2-(2-nitrobenzylamino) propanediol also did not evoke responses. The dihydrosphingosine current had a reversal potential of -11+/-3 mV (n = 11), and was carried by two distinct Cl- and cation currents which were reduced by 85% and about 20% following replacement of monovalent cations with N-methyl-D-glucamine or application of the Cl- channel blocker niflumic acid (10 microM) respectively. The responses to photoreleased dihydrosphingosine were inhibited by intracellular application of 20 mM EGTA, 10 microM ryanodine or extracellular application of 10 microM dantrolene, but persisted when Ca2+ free saline was applied to the extracellular environment. Intracellular application of uncaged dihydrosphingosine evoked responses which were attenuated by photolysis of the caged Ca2+ chelator Diazo-2. Experiments also suggested that extracellular application of dihydrosphingosine can activate membrane conductances. We conclude that dihydrosphingosine directly or indirectly mobilises Ca2+ from ryanodine-sensitive intracellular stores in cultured sensory neurones.

Abstract 15280: Comparisons of Action Potentials and Ionic Currents Between Neonatal Rat Ventricular Myocytes and Adult Zebrafish Ventricular Myocytes

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Adonis Z Wu ◽  
Shien-Fong Lin ◽  
Sheng-Nan Wu
Keyword(s):  
Electrical Properties ◽  
Ventricular Myocytes ◽  
Ionic Currents ◽  
Firing Frequency ◽  
Neonatal Rat ◽  
Adult Zebrafish ◽  
Patch Clamp Technique ◽  
Action Current ◽  
Rat Ventricular Myocytes ◽  
Neonatal Rat Ventricular Myocytes

Introduction: Zebrafish heart is established as a model to investigate cardiac electrical abnormalities. However, electrical properties of adult zebrafish cardiomyocytes are not sufficiently characterized. Hypothesis: In this study, by comparing the electrical properties between neonatal rat ventricular myocytes (NRVMs) and adult zebrafish ventricular myocytes (AZVMs), we intended to characterize the action potential (AP), action current (AC) and the properties of Na + current ( I Na ) in AZVMs. Methods: We used patch-clamp technique to characterize the electrical properties, including AP, AC and I Na , in cultured NRVMs and freshly isolated AZVMs. Results: NVRMs showed larger AP amplitude (119±6 vs. 79±4mV, p<.05) but shorter AP duration (APD 90 , 136±11 vs. 213±19 ms, p<.05) than those of AZVMs. The AP duration exhibited marked frequency-dependent alterations in AZVMs. Under the slow pacing rate, early after-depolarizations (EAD) emerged under slow pacing rate with 0.05 Hz. In cell-attached voltage-clamp recordings made from AZVMs, ACs could be elicited by +10 mV steps. As the depolarization step increased to +70 mV, the latency for appearance of ACs was progressively reduced from >123 ms to 9.8 ms. The presence of spontaneous ACs was monitored in spontaneously beating NRVMs and AZVMs. The AC amplitude in NRVMs was larger compared to that in AZVMs (17.3±2.1 vs. 11.6±1.1 pA, p<.05), although firing frequency of AC in NRVMs is higher than in AZVMs (1.13±0.09 vs. 0.38±0.03 Hz, p<.05). The lowering effect of ranolazine, a I Na antagonist, on firing frequency was significantly larger in NRVMs (1.13±0.09 to 0.31±0.02 Hz, p<.05) than in AZVMs (0.38±0.03 to 0.27±0.02 Hz). There was a hyperpolarizing shift of peak I Na in AZVM compared to NRVM. Conclusions: Our results demonstrated major differences in the cellular electrical behavior between AZVMs and NRVMs.

Characterization of a newly found stretch-activated KCa,ATP channel in cultured chick ventricular myocytes

1999 ◽  
Vol 276 (6) ◽  
pp. H1827-H1838 ◽  
Author(s):  
Takashi Kawakubo ◽  
Keiji Naruse ◽  
Tatsuaki Matsubara ◽  
Nigishi Hotta ◽  
Masahiro Sokabe
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Heart Cells ◽  
Patch Clamp Technique ◽  
Selective Channel ◽  
New Type ◽  
Inside Out ◽  
Ion Selectivities ◽  
Channel Type

With the use of the patch-clamp technique, five kinds of stretch-activated (SA) ion channels were identified on the basis of their single-channel conductances and ion selectivities in cultured chick ventricular myocytes. Because a high-conductance K+-selective channel predominated among these channels, we concentrated on characterizing its properties mostly using excised inside-out patches. With 145 mM KCl solution in the pipette and the bath, the channel had a conductance of 199.8 ± 8.2 pS ( n = 22). The ion selectivities among K+, Na+, Ca2+, and Cl− as estimated from their permeability ratios were P Na/ P K= 0.03, P Ca/ P K= 0.025, and P Cl/ P K= 0.026. The probability of the channel being open (Po) increased with the Ca2+concentration in the bath ([Ca2+]b; dissociation constant K d = 0.51 μM at +30 mV) and membrane potential (voltage at half-maximal Po= 39.4 mV at 0.35 μM [Ca2+]b). The channel was blocked by gadolinium, tetraethylammonium, and charybdotoxin from the extracellular surface and, consequently, was identified as a Ca2+-activated K+(KCa) channel type. The channel was also reversibly activated by ATP applied to the intracellular surface ( K d = 0.74 mM at 0.10 μM [Ca2+]bat +30 mV). From these data taken together, we concluded that the channel is a new type of KCachannel that could be designated as an “SA KCa,ATP channel.” To our knowledge, this is the first report of KCa channel in heart cells.

Glycine regulation of synaptic NMDA receptors in hippocampal neurons

1996 ◽  
Vol 76 (5) ◽  
pp. 3415-3424 ◽  
Author(s):  
K. S. Wilcox ◽  
R. M. Fitzsimonds ◽  
B. Johnson ◽  
M. A. Dichter
Keyword(s):  
Nmda Receptor ◽  
Nmda Receptors ◽  
Hippocampal Neurons ◽  
Time Course ◽  
Hippocampal Slices ◽  
Maximal Effect ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Dissociated Cell Culture ◽  
Cultured Hippocampal Neurons

1. Although glycine has been identified as a required coagonist with glutamate at N-methyl-D-aspartate (NMDA) receptors, the understanding of glycine's role in excitatory synaptic neurotransmission is quite limited. In the present study, we used the whole cell patch-clamp technique to examine the ability of glycine to regulate current flow through synaptic NMDA receptors at excitatory synapses between cultured hippocampal neurons and in acutely isolated hippocampal slices. 2. These studies demonstrate that the glycine modulatory site on the synaptic NMDA receptor is not saturated under baseline conditions and that increased glycine concentrations can markedly increased NMDA-receptor-mediated excitatory postsynaptic currents (EPSCs) in hippocampal neurons in both dissociated cell culture and in slice. Saturation of the maximal effect of glycine takes place at different concentrations for different cells in culture, suggesting the presence of heterogenous NMDA receptor subunit compositions. 3. Bath-applied glycine had no effect on the time course of EPSCs in either brain slice or culture, indicating that desensitization of the NMDA receptor is not prevented by glycine over the time course of an EPSC. 4. When extracellular glycine concentration is high, all miniature EPSCs recorded in the cultured hippocampal neurons contained NMDA components, indicating that segregation of non-NMDA receptors at individual synaptic boutons does not occur.

scholarly journals Bupivacaine inhibits a small conductance calcium‐activated potassium type 2 channel in human embryonic kidney 293 cells

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Hongfei Chen ◽  
Zhousheng Jin ◽  
Fangfang Xia ◽  
Zhijian Fu
Keyword(s):  
Free Calcium ◽  
Heart Cells ◽  
Dependent Manner ◽  
Human Embryonic Kidney ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
293 Cells ◽  
Ic50 Value ◽  
Small Conductance

Abstract Background Bupivacaine blocks many ion channels in the heart muscle, causing severe cardiotoxicity. Small-conductance calcium-activated potassium type 2 channels (SK2 channels) are widely distributed in the heart cells and are involved in relevant physiological functions. However, whether bupivacaine can inhibit SK2 channels is still unclear. This study investigated the effect of bupivacaine on SK2 channels. Methods The SK2 channel gene was transfected into human embryonic kidney 293 cells (HEK-293 cells) with Lipofectamine 2000. The whole-cell patch-clamp technique was used to examine the effect of bupivacaine on SK2 channels. The concentration–response relationship of bupivacaine for inhibiting SK2 currents (0 mV) was fitted to a Hill equation, and the half-maximal inhibitory concentration (IC50) value was determined. Results Bupivacaine inhibited the SK2 channels reversibly in a dose-dependent manner. The IC50 value of bupivacaine, ropivacaine, and lidocaine on SK2 currents was 16.5, 46.5, and 77.8µM, respectively. The degree of SK2 current inhibition by bupivacaine depended on the intracellular concentration of free calcium. Conclusions The results of this study suggested the inhibitory effect of bupivacaine on SK2 channels. Future studies should explore the effects of SK2 on bupivacaine cardiotoxicity.

Abstract 1509: Inducible TBX3 Overexpression As A Tool For Biopacemaker Engineering

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Gerard J Boink ◽  
Martijn L Bakker ◽  
Arie O Verkerk ◽  
Diane Bakker ◽  
Jacques M de Bakker ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Heart Development ◽  
Single Gene ◽  
Neonatal Rat ◽  
Pcr Analysis ◽  
Patch Clamp Technique ◽  
Impulse Propagation ◽  
Transfer Strategies ◽  
Impulse Formation

Introduction: Currently constructed biopacemakers based on single gene transfer strategies function suboptimally, with periods of slow heart rates and instabilities during rest. In the sinoatrial node (SAN), the dominant native pacemaker, multiple genes are required for proper impulse formation and impulse propagation. TBX3 is an important regulator of the SAN gene program during heart development. We examined the effects of inducible TBX3 overexpression in adult hearts and in vitro we explored whether lentiviral TBX3 overexpression may be used in biopacemaker engineering. Methods: In vivo atrial and ventricular expression levels of the connexin isoforms Cx43 and Cx40 (impulse propagation) and SCN5A were studied in mice with tamoxifen inducible overexpression of TBX3 using quantitative PCR analysis. Single neonatal rat cardiac myocytes were transduced with TBX3 expressing lentivirus to analyze the effects of TBX3 on action potentials and membrane currents (impulse formation) using the perforated patch-clamp technique. Results: In vivo, Cx43, Cx40 and SCN5A, which are not or only moderately expressed in the native SAN, were severely down-regulated to 20%, 15%, and 40%, respectively, by TBX3 (n=12; p<0.01). Single neonatal cardiac myocytes overexpressing TBX3 exhibited faster spontaneous beating rates, along with decreased maximum diastolic potential, inward rectifier potassium current (I K1 ), and fast sodium current (I Na ). These properties are typical of SAN pacemaker cells. Conclusions: TBX3 can act as a strong repressor of the working myocardium gene program in the adult heart. Overexpression of TBX3 might be a useful tool in biopacemaker gene and cell therapy.

Abstract 1855: Transmural Myocardial Repair with Engineered Heart Tissue Grafts

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Daniel Biermann ◽  
Michael Didié ◽  
Bijoy Chandapillai Karikkineth ◽  
Claudia Lange ◽  
Thomas Eschenhagen ◽  
...  
Keyword(s):  
Left Ventricle ◽  
Heart Tissue ◽  
Laser Scanning Microscopy ◽  
Neonatal Rat ◽  
Heart Weight ◽  
Heart Cells ◽  
Type I ◽  
Myocardial Repair ◽  
Engineered Heart Tissue ◽  
Tissue Grafts

Engineered Heart Tissue (EHT) can be utilized to partially repair infarcted myocardium in rats. Here, we investigated the feasibility of EHT-grafts as transmural wall replacement in a heterotopic transplantation model. Methods: EHTs (diameter: 15 mm, thickness: 1– 4 mm) were generated from 12.5 ×10 6 neonatal rat heart cells, collagen type I, and matrigel. Similarly, non-contractile constructs were generated from rat cardiac fibroblasts (FB) and mesenchymal stem cells (MSC). Grafts were surgically inserted into large transmural defects (diameter: 6 mm) in the left ventricle of explanted donor hearts. Subsequently, “treated” hearts were transplanted into weight-matched (308±12 g; n=14), immune suppressed (cyclosporine, azathioprine, prednisolone) Wistar rats in heterotopic position. All transmural defects were also covered with an aortic patch to prevent bleeding from the ventricles. Sham surgeries included aortic patch implantations only. Heterotopic hearts were harvested after 28 days and subjected to morphological analyses by confocal laser scanning microscopy (CLSM). Results: Heart transplant weight at the time of implantation was 1.1±0.02 g (n=14). Heterotopic heart weight increased substantially in Sham (2.4±0.3 g, n=3) and FB-graft (2.1±0.1 g, n=3) animals, whereas MSC- (1.7±0.2 g, n=4) and EHT-graft (1.3±0.1 g, n=4; p<0.05 vs. Sham) animals showed a smaller or no increase in weight, respectively. EHT grafts remained contractile throughout the observation period. CLSM revealed that EHT-grafts established oriented muscle bundles (actin and actinin staining) inside the transmural defects and were strongly vascularized (CD31 and smooth muscle actin staining; lectin labeling) leading to partial reconstitution of the myocardial continuity. This was not observed in animals with FB- and MSC-grafts. However, MSC-grafts, but not FB-grafts, contained newly formed vessels with a markedly larger diameter than observed in EHT-grafts (21±6 vs. 5±0.7 μm; p<0.05). Conclusion: EHTs can be utilized as myocardial tissue grafts to reconstruct and prevent pathological enlargement of the left ventricle. This study constitutes a first step to establish a novel transmural myocardial repair technology involving fully bioengineered heart muscle.

Determination of impact of Azadirachta indica L. leaves on adults and eggs of Callosobruchus maculatus Fabricius, the largest predator of stored cowpea (Vigna unguiculata Walp) by an applicable method by farmers

2015 ◽  
Vol 4 (5) ◽  
pp. 215-221
Author(s):  
Ablaye Faye ◽  
Malick Sarr ◽  
Abdoulaye Samb ◽  
Cheikh Thiaw ◽  
Mbacké Sembène
Keyword(s):  
Mortality Rate ◽  
Callosobruchus Maculatus ◽  
Maximum Effect ◽  
Maximal Effect ◽  
Cowpea Weevil ◽  
Lethal Effects ◽  
Neem Leaves ◽  
Leaf Powder ◽  
Higher Doses

  The effect of neem leaves has been tested in the laboratory on eggs and adults of cowpea weevil (C. maculatus). Different formulations of this plant were applied to these forms of C. maculatus Fab. Grinding fresh contact sheets induced significant lethal effects from 96.12% to 100% on eggs; whereas 100% of mortality was recorded at the end of eight days of applica-tion to three adults with higher doses. Fumigation on turn proved less effec-tive than contact on eggs. It induced a maximal effect of 95.73% mortality with the larger dose (D4: 0.02912g/cm3). On adults, we recorded highest mortality (100%) from the 7th day of the show with the highest dose. The aqueous extract of neem leaf powder was less effective than all other formu-lations on the eggs as well as adults of this insect; with a higher mortality rate (74.99%) observed on the eggs with the application of the concentration C2. On adults we recorded a maximum effect (100% mortality) from the 13th day of the application with the highest concentration (C1). These mortalities would be related to the support of several active molecules contained in neem as established in literature.

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