布比卡因对KG1a细胞的中电导钙激活的钾通道的调控作用
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摘要
目的:探讨局部麻醉药布比卡因(Bupivacaine)对中电导钙激活的钾通道(IKCa)的调控作用。方法:体外培养KG1a细胞,PCR检测IKCa基因KCNN4和小电导钙激活的钾通道基因KCNN1-3的表达,全细胞膜片钳方法检测游离Ca2+浓度为1mmol/L的电极内液条件下,破膜前后钾电流变化,并分别观察bupivacaine和IKCa阻断剂Clotrimazole对其的影响。结果:KG1a细胞表达KCNN4mRNA,但不表达KCNN1-3基因;在全细胞记录模式下,刚破膜时记录到微弱钾电流,3min后电流幅度明显增大,同时细胞膜电位由(-22.4±2.3)mV超极化至(-64.3±3.7)mV(P<0.01);IKCa阻断剂Clotrimazole(1mmol/L)作用后,最大电流下降至对照的(34±5)%(P<0.05);bupivacaine(1mmol/L)作用后的电流下降至对照的(28±5)%(P<0.05),该抑制作用可以部分被洗脱。挥发性麻醉剂halothane作用后的KCa电流下降至对照的(30±4)%(P<0.05),但BKCa通道抑制剂四乙铵(TEA,1mmol/L)对KCa无明显抑制作用(P>0.05)。结论:IKCa电流是KG1a细胞的主要KCa电流成分,bupivacaine可以显著抑制IKCa电流。
Objective:To investigate the effect of local anesthetic bupivacaine on the activity of intermediate conductance Ca2+-activated K+channels(IKCa)of KG1 acells.Methods:mRNA expression of IKCa gene KCNN4,and small conductance K+channels(SKCa)gene KCNN1-3 on KG1 acells was determined with PCR,and whole-cell patch-clamp technique was used to record KCa current elicited by 1 mmol/L free Ca2+-containing pipette solution in KG1 acells.Results:PCR experiment showed that KG1 acells expressed only mRNA of KCNN4,but not KCNN1-3.Immediately after the rupture of cell membrane,we recorded a small current,and after diluted with 1 mmol/L free Ca2+-containing pipette solution for 3 min,a robust current was induced,with a reversal potential about-70 mV.This current could be inhibited by 1 mmol/L Clotrimazole,an IKCa blocker,but not by BKCa blocker TEA.The membrane potential of KG1 acells was also hyperpolarized from(-22.4±2.3)mV to(-64.3 ± 3.7)mV(P<0.01)after dilution with1 mmol/L free Ca2+-containing pipette solution.Bupivacaine(1 mmol/L)inhibited this current to(28±5)% of control(P<0.05).In addition,this current was also significantly inhibited by volatile anesthetic halothane at 1 mmol/L.Conclusion:IKCa is the major KCa channel in KG1 a cells,and bupivacaine can potently inhibit IKCa channels on KG1 acells.
Objective:To investigate the effect of local anesthetic bupivacaine on the activity of intermediate conductance Ca2+-activated K+channels(IKCa)of KG1 acells.Methods:mRNA expression of IKCa gene KCNN4,and small conductance K+channels(SKCa)gene KCNN1-3 on KG1 acells was determined with PCR,and whole-cell patch-clamp technique was used to record KCa current elicited by 1 mmol/L free Ca2+-containing pipette solution in KG1 acells.Results:PCR experiment showed that KG1 acells expressed only mRNA of KCNN4,but not KCNN1-3.Immediately after the rupture of cell membrane,we recorded a small current,and after diluted with 1 mmol/L free Ca2+-containing pipette solution for 3 min,a robust current was induced,with a reversal potential about-70 mV.This current could be inhibited by 1 mmol/L Clotrimazole,an IKCa blocker,but not by BKCa blocker TEA.The membrane potential of KG1 acells was also hyperpolarized from(-22.4±2.3)mV to(-64.3 ± 3.7)mV(P<0.01)after dilution with1 mmol/L free Ca2+-containing pipette solution.Bupivacaine(1 mmol/L)inhibited this current to(28±5)% of control(P<0.05).In addition,this current was also significantly inhibited by volatile anesthetic halothane at 1 mmol/L.Conclusion:IKCa is the major KCa channel in KG1 a cells,and bupivacaine can potently inhibit IKCa channels on KG1 acells.
引文
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[11]Tamanai-Shacoori Z,Shacoori V,Vo VJ,et al.Sufentanil modifies the antibacterial activity of bupivacaine and ropivacaine[J].Can J Anaesth,2004,51(9):911-914.
[12]Barnes EA,Lee L,Barnes SL,et al.β1-Subunit of the calcium-sensitive potassium channel modulates the pulmonary vascular smooth muscle cell response to hypoxia[J].Am J Physiol Lung Cell Mol Physiol,2018,15(2):L265-L275.
[13]Hashiguchi-Ikeda M,Namba T,Ishii TM,et al.Halothane inhibits an intermediate conductance Ca2+-activated K+channel by acting at the extracellular side of the ionic pore[J].Anesthesiology,2003,99(6):1 340-1 345.
[14]Nguyen HM,Singh V,Pressly B,et al.Structural insights into the atomistic mechanisms of action of small molecule inhibitors targeting the KCa3.1channel pore[J].Mol Pharmacol,2017,91(4):392-402.
[2]Nakahira K,Oshita K,Itoh M,et al.Clinical concentrations of local anesthetics bupivacaine and lidocaine differentially inhibit human Kir2.x inward rectifier K+channels[J].Anesth Analg,2016,122(4):1 038-1 047.
[3]Shin HW,Soh JS,Kim HZ,et al.The inhibitory effects of bupivacaine,levobupivacaine,and ropivacaine on K2P(two-pore domain potassium)channel TREK-1[J].J Anesth,2014,28(1):81-86.
[4]Kang D,Hogan JO,Kim D.THIK-1(K2P13.1)is a small-conductance background K(+)channel in rat trigeminal ganglion neurons[J].Pflugers Arch,2014,466(7):1 289-1 300.
[5]Kaczmarek LK,Aldrich RW,Chandy KG,et al.International union of basic and clinical pharmacology.C.nomenclature and properties of calcium-activated and sodium-activated potassium channels[J].Pharmacol Rev,2017,69(1):1-11.
[6]Horrigan FT.Perspectives on:conformational coupling in ion channels:conformational coupling in BKpotassium channels[J].J Gen Physiol,2012,140(6):625-634.
[7]Christophersen P,Wulff H.Pharmacological gating modulation of small-and intermediate-conductance Ca(2+)-activated K(+)channels(KCa2.x and KCa3.1)[J].Channels(Austin),2015,9(6):336-343.
[8]Namba T,Ishii TM,Ikeda M,et al.Inhibition of the human intermediate conductance Ca(2+)-activated K(+)channel,hIK1,by volatile anesthetics[J].Eur JPharmacol,2000,395(2):95-101.
[9]Joiner WJ,Wang LY,Tang MD,et al.hSK4,a member of a novel subfamily of calcium-activated potassium channels[J].Proc Natl Acad Sci U S A,1997,94(20):11 013-11 018.
[10]Gungor I,Yilmaz A,Ozturk AM,et al.Bupivacaine and levobupivacaine induce apoptosis in rat chondrocyte cell cultures at ultra-low doses[J].Eur J Orthop Surg Traumatol,2014,24(3):291-295.
[11]Tamanai-Shacoori Z,Shacoori V,Vo VJ,et al.Sufentanil modifies the antibacterial activity of bupivacaine and ropivacaine[J].Can J Anaesth,2004,51(9):911-914.
[12]Barnes EA,Lee L,Barnes SL,et al.β1-Subunit of the calcium-sensitive potassium channel modulates the pulmonary vascular smooth muscle cell response to hypoxia[J].Am J Physiol Lung Cell Mol Physiol,2018,15(2):L265-L275.
[13]Hashiguchi-Ikeda M,Namba T,Ishii TM,et al.Halothane inhibits an intermediate conductance Ca2+-activated K+channel by acting at the extracellular side of the ionic pore[J].Anesthesiology,2003,99(6):1 340-1 345.
[14]Nguyen HM,Singh V,Pressly B,et al.Structural insights into the atomistic mechanisms of action of small molecule inhibitors targeting the KCa3.1channel pore[J].Mol Pharmacol,2017,91(4):392-402.