The Effect Of Different Chemicalstrcture Muscle Relxants On Muscular Adult-type And Fetal-type Nicotinic Acetylcholine Receptors

Doctor candidate: Xuelian Zhao Supervisor: Prof. Xinliang ZhuangMuscle relaxants block neuromuscular transmission by binding competitively to the muscle nicotinic acetylcholine receptor in the post-junctional memberane of the neuromuscular junction. The affect of muscle relaxants was variously for determined by many factors. We can study deeply main influence factor of action of muscle relaxants by researching interaction between muscle relaxants and nicotinic acetylcholine receptors. Objective: Muscle adult-type and fetal-type nicotinic acetylcholine receptors were temporarily heterologously expressed in human embryonic kidney 293 cells(HEK293). We study interaction between rocuronium,vecurnium,atracurium individually and combinably and adult-type, fetal-type nicotinic acetylcholine receptor using whole cell patch clamp technique.Methods: We obtained pcDNA3.1, pcDNA3.1p, pcDNA3.1, pcDNA3.1y, pcDNA3.1 E plasmid cutting cDNA fragments of objectved gene a, p, 8, y, e subunit from prokaryotic plasmid vector and inserting into pcDNA3.1 vectors. pcDNA3.1, pcDNA3.1p, pcDNA3.1, pcDNA3.1, pcDNA3.1 plasmid were transformated into E. coli viruses,then we selected and identified positive recombinant plasmids using inspecting sequence of cDNA fragments of HEK293 cells were cultured in DMEM supplemented with 10% calf serum at 37 in 5%CO2 incubator. HEK293 cells were transfected with pcDNA3.1 vectors. pcDNA3.1 , pcDNA3.1 , pcDNA3.1, pcDNA3.1 or pcDNA3.1 at 2:1:1:1 rate using DMRIE-C Reagent. Peak currents were recorded in HEK293 cells treated by rocuronium,vecuronium , atracurium individually using whole cell patch clamp technique.Drug combinations containing equipotent concentrations of rocuronium and vecuronium or rocuronium and atracurium were tested and dose-response curves were determined.Results: We gained HEK293 cells expressing adult-type or fetal-type nicotinic acetylcholine receptors. Currents were recorded from HEK293 cells which transfectedby pcDNA3.1, pcDNA3.1, pcDNA3.1, pcDNA3.1, pcDNA3.1 using whole cell patch clamp. There were not any current from HEK293 cells which transfected pcDNA3.1 alone and nontransfection. Rovuronium,vecuronium and atracurium competitively blocked nAChR and Y -nAChR of HEK293 cells. IC50 values of rovuronium,vecuronium and atracurium to nACHR were 169.21 12.5M , 8.29 2.65M and 24.23 10.48M respectively; IC50 values of rocuronium, vecuronium and atracurium to nAChR were 8.64 2.65M , 54.97 10.45M and 183.29 39.26M respectively. Isobolographic analysis indicate that to e -nAChR ,the experimental IC50 values for the individual drugs in combination of rocuronium with vecronium or atracurium fell at the below of the 95% confidence interval. The experimental IC50 values for the individual drugs in combination of rocuronium with vecronium to y -nAChR fell in the 95% confidence interval an in combination of rocuronium with atracurium fell at the below of the 95% confidence interval.Conclusion: CD We obtained HEK293 cells ,experimental plate, which expressing adult-type and fetal-type nicotinic acetylcholine receptors respectively.(2) The potency of inhibition to nAChR is vecuronium>atracurium>rocuronium from strength to weak in turn, and to y -nAChR is rocuronium >vecuronium>atracurium in turn;(3) Inffinity of nAChRto rocuronium was least among vecuronium and atracurium. Inffmity of nAChR to rocuronium was most. (4)Isobolographic analyses indicated synergistic interaction for rocuronium combined with vecronium or atracurium to ?-nAChR. Isobolographic analyses indicated additional interaction for rocuronium combined with vecronium to y -nAChR and synergistic interaction for rocuronium combined with atracurium to y -nAChR as well.