scholarly journals Bupivacaine inhibits a small conductance calcium-activated potassium type 2 channel (SK2) in HEK 293 cells

2019 ◽  
Author(s):  
Hongfei Chen ◽  
Fangfang Xia ◽  
Zhousheng Jin ◽  
Yuting He ◽  
Zhengjie Chen ◽  
...  
Keyword(s):  
Inhibitory Effect ◽  
Heart Cells ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Ic50 Value ◽  
Small Conductance

Abstract Background: Bupivacaine blocks many ion channels in the heart muscle, which could cause 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 unknown. 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 study the effect of bupivacaine on SK2 channels. The inhibitory effect of various concentrations of bupivacaine on SK2 currents exhibited a non-linear relation, 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 the SK2 current was 133.7, 189.3, and 885.8 µM, respectively. The degree of SK2 current inhibition by bupivacaine was dependent on the intracellular concentration of free calcium. Conclusions: The results of this study suggested a new inhibitory effect of bupivacaine on SK2 channels. Future studies should be concerned with the effects of SK2 on bupivacaine cardiotoxicity. Keywords: Bupivacaine, SK2 channel, inhibition, cardiotoxicity, HEK 293.

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.

scholarly journals Bupivacaine inhibits a small-conductance calcium-activated potassium type 2 channel (SK2) in HEK293 cells

2020 ◽  
Author(s):  
Hongfei Chen ◽  
Fangfang Xia ◽  
Zhousheng Jin ◽  
Zhijian Fu
Keyword(s):  
Hek293 Cells ◽  
Free Calcium ◽  
Heart Cells ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
293 Cells ◽  
Ic50 Value ◽  
Small Conductance

Abstract Background: Bupivacaine blocks many ion channels in the heart muscle, which could cause 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 unknown. 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 study the effect of bupivacaine on SK2 channels. Concentration-response relationship of bupivacaine for inhibiting SK2 current (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 the SK2 current was 16.5, 46.5, and 77.8 µM, respectively. The degree of SK2 current inhibition by bupivacaine was dependent on the intracellular concentration of free calcium.Conclusions: The results of this study suggested a new inhibitory effect of bupivacaine on SK2 channels. Future studies should be concerned with the effects of SK2 on bupivacaine cardiotoxicity.

scholarly journals Inhibition of Small-Conductance, Ca2+-Activated K+ Current by Ondansetron

2021 ◽  
Vol 12 ◽  
Author(s):  
Shuai Guo ◽  
Zhenhui Chen ◽  
Peng-Sheng Chen ◽  
Michael Rubart
Keyword(s):  
Inhibitory Effect ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Inwardly Rectifying ◽  
Small Conductance

Background: Small-conductance Ca2+-activated K+ channels (SK channels) have been proposed as antiarrhythmic targets for the treatment of atrial fibrillation. We previously demonstrated that the 5-HT3 receptor antagonist ondansetron inhibits heterologously expressed, human SK2 (hSK2) currents as well as native cardiac SK currents in a physiological extra-/intracellular [K+] gradient at therapeutic (i.e., sub-micromolar) concentrations. A recent study, using symmetrical [K+] conditions, challenged this result. The goal of the present study was to revisit the inhibitory effect of ondansetron on hSK2-mediated currents in symmetrical [K+] conditions.Experimental Approach: The whole-cell patch clamp technique was used to investigate the effects of ondansetron and apamin on hSK2-mediated currents expressed in HEK 293 cells. Currents were measured in symmetrical [K+] conditions in the presence of 100 nM [Ca2+]o.Results: Expression of hSK2 produced inwardly rectifying whole-cell currents in the presence of 400 nM free cytosolic Ca2+. Ondansetron inhibited whole-cell hSK2 currents with IC50 values of 154 and 113 nM at −80 and 40 mV, respectively. Macroscopic current inhibited by ondansetron and current inhibited by apamin exhibited inwardly rectifying current-voltage relationships with similar reversal potentials (apamin, ∼5 mV and ondansetron, ∼2 mV). Ondansetron (1 μM) in the continuing presence of apamin (100 nM) had no effect on hSK2-mediated whole-cell currents. Wild-type HEK 293 cells did not express ondansetron- or apamin-sensitive currents.Conclusion: Ondansetron in sub-micromolar concentrations inhibits hSK2 currents even under altered ionic conditions.

Ablation of small conductance calcium-activated potassium type-2 channel (SK2) delays occurrence of bupivacaine-induced cardiotoxicity in isolated mouse hearts

2020 ◽  
pp. 096032712095810
Author(s):  
H Chen ◽  
F Xia ◽  
X Chen ◽  
Y Cai ◽  
Z Jin
Keyword(s):  
Rate Pressure Product ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Severe Arrhythmia ◽  
293 Cells ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent ◽  
Small Conductance

Bupivacaine is frequently used for conducting regional anesthesia. When accidentally injected or excessively absorbed into circulation, bupivacaine can induce severe arrhythmia and potentially lead to cardiac arrest. The specific mechanisms underlying this cardiotoxicity, however, remain to be clarified. We transfected HEK-293 cells to express the small conductance calcium-activated potassium type-2 channel (SK2), and used a whole-cell patch clamp method in order to explore how bupivacaine affected these channels. We subsequently used SK2 knockout mice to explore the relevance of SK2 channels in bupivacaine-induced cardiotoxicity in isolating mouse hearts, mounting them on a Langendorff apparatus, and perfusing them with bupivacaine. Using this system, arrhythmia, asystole, and cardiac functions were monitored. We observed dose-dependent inhibition of SK2 channels by bupivacaine: half-maximal inhibitory concentration (IC50) value = 18.6 μM (95% CI 10.8–32.1). When SK2 knockout (SK2 −/−) or wild-type (WT) mice were perfused with Krebs-Henseleit buffer (KHB), we did not observe any instances of arrhythmia. When SK2 −/− mice or WT were perfused with KHB containing bupivacaine (40 μM), the time to arrhythmia (Tarrhythmia) and time to asystole (Tasystole) were both significantly longer in SK2 −/− mice relative to WT mice ( P < 0.001). Similarly, SK2 −/− mice exhibited a significantly longer time to 25%, 50%, and 75% reductions in heart rate (HR) and rate-pressure product (RPP) relative to WT mice following bupivacaine perfusion ( P < 0.001). These results reveal that bupivacaine was able to mediate a dose-dependent inhibition of SK2 channels in HEK-293 cells, and deletion of SK2 channels can delay bupivacaine-induced cardiotoxicity in isolated mouse hearts.

Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

2005 ◽  
Vol 103 (6) ◽  
pp. 1156-1166 ◽  
Author(s):  
Kevin J. Gingrich ◽  
Son Tran ◽  
Igor M. Nikonorov ◽  
Thomas J. Blanck
Keyword(s):  
Charge Transfer ◽  
Calcium Channels ◽  
Dependent Manner ◽  
Current Time ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Dependent Inactivation ◽  
Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

scholarly journals Polyphenolic Compounds and Antioxidant Activities of the Leaves of Glochidion hypoleucum

2015 ◽  
Vol 10 (3) ◽  
pp. 1934578X1501000
Author(s):  
Natthinee Anantachoke ◽  
Worawan Kitphati ◽  
Supachoke Mangmool ◽  
Nuntavan Bunyapraphatsara
Keyword(s):  
Gallic Acid ◽  
Methanol Extract ◽  
Radical Scavenging ◽  
Dependent Manner ◽  
Polyphenol Compounds ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Chemical Structures ◽  
293 Cells ◽  
Crude Methanol Extract

Bioassay-guided fractionation of the methanol extract of Glochidion hypoleucum (Miq.) Boerl leaves led to the isolation of five polyphenol compounds, methyl gallate, gallic acid, apigenin-8- C-β-D-glucopyranoside (vitexin), luteolin-8- C-β-D-glucopyranoside (orientin), and luteolin-6- C-β-D-glucopyranoside (isoorientin). The chemical structures of the isolated compounds were determined using spectroscopic (NMR, UV-Vis, IR) and mass spectrometric techniques. The antioxidative properties of the methanol extract and isolated polyphenols were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) for radical scavenging activity and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) to measure the level of reactive oxygen species (ROS). With the exception of vitexin, the crude methanol extract and the polyphenol compounds inhibited DPPH radicals with IC50 values ranging from 2.46 ± 0.05 to 40.0 ± 0.3 μg/mL. In addition, the crude methanol extract attenuated H2O2-induced intracellular ROS production in a dose-dependent manner in HEK-293 cells. Gallic acid and isoorientin significantly reduced the ROS level in HEK-293 cells at a concentration of 20 μM.

SPI-0211 activates T84 cell chloride transport and recombinant human ClC-2 chloride currents

2004 ◽  
Vol 287 (5) ◽  
pp. C1173-C1183 ◽  
Author(s):  
John Cuppoletti ◽  
Danuta H. Malinowska ◽  
Kirti P. Tewari ◽  
Qiu-ju Li ◽  
Ann M. Sherry ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Chloride Transport ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Adult Rabbit ◽  
Dependent Manner ◽  
T84 Cells ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

The purpose of this study was to determine the mechanism of action of SPI-0211 (lubiprostone), a novel bicyclic fatty acid in development for the treatment of bowel dysfunction. Adult rabbit intestine was shown to contain mRNA for ClC-2 using RT-PCR, Northern blot analysis, and in situ hybridization. T84 cells grown to confluence on permeable supports were shown to express ClC-2 channel protein in the apical membrane. SPI-0211 increased electrogenic Cl− transport across the apical membrane of T84 cells, with an EC50 of ∼18 nM measured by short-circuit current ( Isc) after permeabilization of the basolateral membrane with nystatin. SPI-0211 effects on Cl− currents were also measured by whole cell patch clamp using the human embryonic kidney (HEK)-293 cell line stably transfected with either recombinant human ClC-2 or recombinant human cystic fibrosis transmembrane regulator (CFTR). In these studies, SPI-0211 activated ClC-2 Cl− currents in a concentration-dependent manner, with an EC50 of ∼17 nM, and had no effect in nontransfected HEK-293 cells. In contrast, SPI-0211 had no effect on CFTR Cl− channel currents measured in CFTR-transfected HEK-293 cells. Activation of ClC-2 by SPI-0211 was independent of PKA. Together, these studies demonstrate that SPI-0211 is a potent activator of ClC-2 Cl− channels and suggest a physiologically relevant role for ClC-2 Cl− channels in intestinal Cl− transport after SPI-0211 administration.

scholarly journals Calcium-sensing receptors control CYP27B1-luciferase expression. Transcriptional and post-transcriptional mechanisms

2021 ◽  
Author(s):  
Alice Huang ◽  
Lenah Binmahfouz ◽  
Dale P Hancock ◽  
Paul H Anderson ◽  
Donald T Ward ◽  
...  
Keyword(s):  
Fold Increase ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Renilla Luciferase ◽  
Luciferase Expression ◽  
Mrna Levels ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Calcium Sensing ◽  
293 Cells

Abstract 25-hydroxyvitamin D 1α-hydroxylase (encoded by CYP27B1), which catalyses the synthesis of 1,25-dihydroxyvitamin D3, is subject to negative or positive modulation by extracellular Ca 2+ (Ca 2+o) depending on the tissue. However, the Ca 2+ sensors and underlying mechanisms are unidentified. We tested whether calcium-sensing receptors (CaSRs) mediate Ca 2+o-dependent control of 1α-hydroxylase using HEK-293 cells stably expressing the CaSR (HEK-CaSR cells). In HEK-CaSR cells, but not control HEK-293 cells, co-transfected with reporter genes for CYP27B1-Photinus pyralis (firefly) luciferase and control Renilla luciferase, an increase in Ca 2+o from 0.5 to 3.0 mM induced a 2-3 fold increase in firefly-luciferase activity as well as mRNA and protein levels. Surprisingly, firefly-luciferase was specifically suppressed at Ca 2+o ≥ 5.0 mM, demonstrating biphasic Ca 2+o control. Both phases were mediated by CaSRs as revealed by positive and negative modulators. However, Ca 2+o induced simple monotonic increases in firefly-luciferase and endogenous CYP27B1 mRNA levels, indicating that the inhibitory effect of high Ca 2+o was post-transcriptional. Studies with inhibitors and the CaSR C-terminal mutant T888A identified roles for PKC, phosphorylation of T888, and ERK1/2 in high Ca 2+o-dependent suppression of firefly-luciferase. Blockade of both PKC and ERK1/2 abolished Ca 2+o-stimulated firefly-luciferase, demonstrating that either PKC or ERK1/2 is sufficient to stimulate the CYP27B1 promoter. A key CCAAT box (–74 bp to –68 bp), which is regulated downstream of PKC and ERK1/2 was required for both basal transcription and Ca 2+o-mediated transcriptional upregulation. The CaSR mediates Ca 2+o-dependent transcriptional upregulation of 1α-hydroxylase and an additional CaSR-mediated mechanism is identified by which Ca 2+o can promote luciferase and possibly 1α-hydroxylase breakdown.

Type 2 IP3 receptors are involved in uranyl acetate induced apoptosis in HEK 293 cells

Toxicology ◽  
2009 ◽  
Vol 262 (1) ◽  
pp. 73-79 ◽  
Author(s):  
Juraj Kopacek ◽  
Karol Ondrias ◽  
Barbora Sedlakova ◽  
Jana Tomaskova ◽  
Lucia Zahradnikova ◽  
...  
Keyword(s):  
Uranyl Acetate ◽  
Ip3 Receptors ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Induced Apoptosis
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