Retigabine Reverses The Inhibition Of KCNQ2/Q3Channel Currents And Seizures Induced By Local Anesthetic

Part1Chloroprocaine impacts on KCNQ2/Q3channel currents andretigabine reverse the the effect of chloroprocainObjective: To characterize Chloroprocaine effects on KCNQ2/Q3chan-nels and to investigate whether retigabine reverse the effects of chloroprocai-ne.Methods: Human embryonic kidney （HEK293） cells, as an expressionsystem, were transfected with KCNQ2, KCNQ3cDNAs by usinglipofectamine. The effects of chloroprocaine and retigabine on KCNQ2/Q3currents was observed by using the method of whole cell patch clamp.Theexperiment was performed in three steps.The first step: To investigate the effect of the different concentration ofchloroprocaine on KCNQ2/Q3channel current under different clampingvoltage. According to the different concentration of chloroprocaine,chloroprocaine were divided into4groups,0mmol/L,1mmol/L,10mmol/L,100mmol/L,0mmol/L as a control group （n=11）. Each group were clampedby step depolarization to-40mV,0mV and40mV from a holding potential of-80mV. Record the KCNQ2/Q3channel current with different concentrationof chloroprocaine and under different clamping voltage.The second step: To study the effect of chloroprocaine on the kinetics ofKCNQ2/Q3currents. Cells were clamped by a train of voltage from-80mV to+30mV in a10mV interval from a holding potential of-80mV,thenrepolarized to-60mV.10mmol/L chloroprocaine was given by externalperfusion,0mmol/L chloroprocaine as a control, each observed five samples.Different test potentials were normalized and fitted by Boltzmann function,and fitted KCNQ2/Q3channel I-V curve. The activation and deactivationcurrents were both fitted to a single exponential function. The third step: To explore the effect of100mmol/L chloroprocaine onKCNQ2/Q3channel currents in the presence of retigabine.Cells were clampedby a step depolarization to0mV from a holding potential of-80mV,thenrepolarized to-80mV.100mmol/L chloroprocaine or containing10μmol/Lretigabine was given by external perfusion,0mmol/L chloroprocaine as acontrol, each observed five samples. Recorded KCNQ2/Q3channel currentsunder0mV.Results:1Compared with control group, there was significant differ--ence in inhibition of KCNQ2/Q3channel currents in group C₁, group C₂,andgroup C₃respectively （P<0.05） under+40mV. Compared with controlgroup, under0mV the inhibition of KCNQ2/Q3channel currents weresignificantly higher in group C₁, group C₂,and group C₃respectively（P<0.05）. Compared with control group, under-40mV the inhibition ofKCNQ2/Q3channel currents were significantly higher in group C₁,group C₂,and group C₃respectively （P<0.05）.2The time constant for the currentactivation at0mV was26.9±3.9ms for the control and36.4±4.1msfor chloroprocaine, respectively （n=5, P<0.05）. And for the currentdeactivation, the time constant was71.3±4.7ms for the control and65.0±6.1ms for chloroprocaine, respectively.（n=5, P<0.05）.310mmol.L-1chloroprocaine shifted the voltage for half-maximal activation (V_1/2) ofKCNQ2/Q3currents from （-36.8±1.7） mV to （-26.1±1.3） mV （P<0.05）.4Chloroprocaine （100mmol/L） significantly reduced KCNQ2/Q3channel currents by72.9±3.0%. However, the inhibitory effect ofchloroprocaine was reduced to4.9±0.1%in the presence of retigabine（10μmol/L）（n=5, P<0.05）.Conclusions:1Chloroprocaine concentration dependently and voltage-dependently suppresses KCNQ2/Q3channel current.2Chloroprocaine slowedchannel activation and accelerated deactivation kinetics as well as shiftedrightward the voltage-dependent activation curves.3Retigabine is able toreverse the inhibition of Chloroprocaine on KCNQ2/Q3channel currents. Part ⅡEffect of retigabine on median lethal dose and median effectivedose of bupivacaine and chloroprocaine for induction of convulsionin miceObjective: To investigate the effect of KCNQ2/Q3channel openerretigabine on the median letal dose (LD₅₀) and median effective dose (ED₅₀)of bupivacaine and chloroprocaine for induction of convulsion in mice and therelationship between KCNQ2/Q3channels and the toxicity of localanesthetics.Methods: Pathogen-free female Kunming mice, weighting20³0g, wereused in the study. The experiment was performed in two parts. Part Ⅰ：Onehunder and forty mice were randomly divided into2groups （n=70each）:control group （group C） and retigabine group （group R）. The C and R groupswere further divided into7subgroups depending on the dose of chlorprocaine（C+L₁, C+L₂, C+L₃,C+L₄, C+L₅, C+L₆and C+L₇groups, and R+L₁, R+L₂,R+L₃, R+L₄, R+L₅, R+L₆, and R+L₇groups，n=10each）. In groups C and R,0.9%normal saline0.005ml/g and retigbine20mg/kg were injectedintraperitoneally respectively, and chlorprocaine was injected intraperitoneally20min later. The dose of chlorprocaine were150.0,172.5,198.4,228.2,262.4,301.8, and346.1mg/kg in C+L₁,C+L₂, C+L₃, C+L₄, C+L₅, C+L₆, and C+L₇groups respectively, and198.4,228.2,262.4,301.8,346.1,398.0and457.7mg/kg in R+L₁, R+L₂, R+L₃, R+L₄, R+L₅, R+L₆and R+L₇groupsrespectively. PartⅡ One hundred mice were randomly divided into2groups（n=50each）: control group （group C） and retigabine group （group R）. The Cand R groups were further divided into5subgroups with different doses ofbupivacaine （C+B₁, C+B₂, C+B₃, C+B₄and C+B₅groups, and R+B₁, R+B₂,R+B₃, R+B₄and R+B₅groups, n=10each）.In groups C and R,0.9%normalsaline0.005ml/g and retigbine20mg/kg were injected intraperitoneallyrespectively, and bupivacaine was injected intraperitoneally20min later. The dose of bupivacaine were37.8,43.5,50.0,57.5and66.1mg/kg inC+B₁,C+B₂,C+B₃, C+B₄and C+B₅groups respectively, and50.0,57.5,66.1,76.0and87.4mg/kg in R+B₁,R+B₂,R+B₃,R+B₄and R+B₅groups, respectively. The ED₅₀and95%confidence interval （95%CI）, LD₅₀and95%confidence interval （95%CI） of chloroprocaine and bupivacaine forinduction of convulsion were calculated by Probit analysis.Results: The ED₅₀（95%CI） of chloroprocaine was165.3（155.8¹75.0）mg/kg, and the LD₅₀（95%CI） of chloroprocaine was316.5（290.4³54.3）mg/kg；the ED₅₀（95%CI） of bupivacaine was41.1（36.7⁴4.5）mg/kg，andthe LD₅₀（95%CI） of bupivacaine was58.9（54.7⁶4.4）mg/kg in C group.The ED₅₀（95%CI） of chloroprocaine was212.4（200.2²24.3）mg/kg, andthe LD₅₀（95%CI） of chloroprocaine was402.8（371.0⁴47.4）mg/kg；theED₅₀（95%CI） of bupivacaine was51.5（45.1⁵6.0）mg/kg，and the LD₅₀（95%CI） of bupivacaine was77.8（71.8⁸4.9）mg/kg in R group. Copmaredwith group C, the ED₅₀and LD₅₀of chloroprocaine and bupivacaine forinduction of convulsion were significantly increased in group R （P<0.01）.Conclusions:1KCNQ2/Q3channel opener retigabine can significantlyincrease the ED₅₀of chloroprocaine and bupivacaine for induction ofconvulsion and reduce convulsion induced by bupivacaine and chloroprocainein mice, indicating that the neurotoxicity of local anesthetics is related toinhibition of KCNQ2/Q3channels.2KCNQ2/Q3channel opener retigabinecan significantly increase the LD₅₀of chloroprocaine and bupivacaine，reduce mortality of bupivacaine and chloroprocaine in mice. PartⅢ The anticonvulsant action of retigabine onβ-band ofelectroencephalogram in convulsion rabbits induced by levobupivacaineObjective: To observe the effect of retigabine on the seizure induced bylevobupivacaine in rabbits, as well as the effect of retigabine onβ-band ofelectroencephalogram in convulsion rabbits. Explore the role of retigabine on local anesthetic induces convulsions.Methods: Twenty rabbits were randomly divided into two groups （n=10each）: control group （group C） and retigabine group （group R）.8ml/（kg×h） of0.5%levobupivacaine were given intravenously in these rabbits until tonicconvulsions occurred and5mg/kg of retigabine in2ml saline wereintravenously delivered to relief the convulsion, or2ml of saline was given ascontrol. Dose of levobupivacaine used for induction of convulsions wascalculated and duration of tonic state was noted. The EEG activity wasprocessed with the power spectral analysis. The percentage of power ofβ-band of EEG sample were obtained at different time after drug injection.Results:1There was no significant difference of the dose of levobup-ivacaine for the induction of convulsions in two groups （P>0.05）. Comparedwith control group, the behavior and EEG of epilepsy of experiment groupwere significantly shorter （P<0.01）.2Compare with the control group, thepercentage of β-band power of EEG decreased significantly （P<0.01） in theretigabine group with1¹5minute after therapy. There was no significantdifference of the percentage ofβ-band power of EEG in the retigabine groupcompare to the control group with20³0minute after therapy（P>0.05）.Compared with the baseline, the percentage of β-band graduallyreduced within30minute after treatment in the control group. Compare withthe baseline, the percentage ofβ-band power of EEG decreased significantly（P<0.01） in the retigabine group with1¹5minute after therapy. Comparedwith the baseline, the percentage of β-band power of EEG increasedsignificantly （P<0.01） in the two groups,respectively.Conclusion: Retigabine can shorten the seizure duration induced bylevobupivacaine. Retigabine has the effect of anti-convulsion induced bylevobupivacaine. In the systemic toxicity induced by levobupivacaine,retigabine could reduce the central nervous system activity and reduce thefrequency of epilepsy wave issuance.