Effect Of Dexmedetomidine Hydrochloride On BK_Ca Of Porcine Coronary Artery Smooth Muscle And Mechanism

Objective:As a highly selective α2 adrenoeeptor(α2AR)agonist,dexmedetomidine has been widely used in perioperative period for its sedative,analgesic,anxiolytic and sympathetic effects and the effect of reducing stress response,decreasing anesthetic dosage and stabilizing hemodynamics.Animal experiments and clinical studies have found that dexmedetomidine have good cardioprotective effect.Large Ca2+-activated potassium channels(BKCa)serve as the most important outward current channels in VSMCs.Previous studies found that clinical dose of dexmedetomidine can relax the porcine coronay artery,BKCa channel inhibitors TEA(10-5 mmol/L)can decrease significantly vasodilation of porcine coronary artery induced by dexmedetomidine,which show BKCa associated with dexmedetomidine-induced dilation.However,it has not been reported that dexmedetomidine activate coronary artery BKCa directly.Therefore,this paper is designed to observe the effect of different concentrations of dexmedetomidine on primary and transfected BKCa using patch clamp technique(inside-out mode),explore dexmedetomidine on BKCa channel kinetics properties.Methods: 1)The proximal blood vessel of porcine coronary artery left anterior descending branch was separated and cutinto small pieces,followed by digesting the tissue into single SMCs via twostep enzyme digestion.With the membrane potential clamped at +40 m V,The cells were placed in symmetric high potassium solution([K+]0:[K+]i=140:140m M),the effect of different concentrations of dexmedetomidine hydrochloride(0,10-9,10-8,10-7,10-6,10-5mol/L)on BKCa was recorded in the inside-out configuration.2)The culture and subculture were conducted using DMEM high glucose medium(containing 10% FBS)in the incubator of 5% CO2 atmosphere.Lipofection(lipofectamine 3000,Invitrogen)was employed to transfect 5 μg of β1plasmids to a 3.5-cm culture dish.The resulting HEK293 cell line stably expressing p CDNA3.1-BKα orα+β1 was used for further experiment.In symmetric high potassium solution,The membrane potential at +40 m V,the effect of different concentrations of dexmedetomidine(0,10-9,10-8,10-7,10-6,10-5 mol/L)on BKCa was recorded using patch clamp technique.Open Probability(NPo),Amplitude(Amp),mean open Time(To)and mean close Time(Tc)of the current obtained was amplified through CEZ-2300 and p CLAMP10.1 software was adopted for data acquisition and analysis.Results: 1.In symmetrical high potassium solution,the BKCa of porcine coronary artery smooth muscle exerted high conductance,voltage dependence and calcium dependence.2.In symmetrical high potassium solution with Ca2+ concentration of 10-7 M,no significant differences in average channel open time and current amplitude of BKCa in porcine coronary artery smooth muscle experiment were observed atevery concentration of dexmedetomidine(P>0.05);with the increase of dexmedetomidine(10-9-10-5 M),Tc was gradually reduced(P<0.05);NPo had no change at lower concentrations of dexmedetomidine(10-9-10-8 M)(P>0.05),while NPo was increased and had dose-dependent(P<0.05)when dexmedetomidine increased between 10-7 and 10-5 M.3.With Ca2+ of 0 M,no significant differences in To,Tc,Amp and NPo between expressing α-and α+β-subunit BKCa(P>0.05);with Ca2+ concentration(10-7 M),compared with expressing α-subunit BKCa,To and NPo were gradually increased(P<0.05),but no significant differences in Tc,Amp of expressing α+β-subunit BKCa and expressing α-subunit BKCa in the HEK293 cells,(P>0.05).4.Compared with group without dexmedetomidine(control group)(P>0.05),the lower concentrations of dexmedetomidine(10-9-10-7 M)did not increase NPo of the expressing α-subunit BKCa channel,while the NPo was gradually elevated and became dose-dependent under higher concentrations of dexmedetomidine(10-6-10-5 M),but no significant differences in To,Tc,Amp(P>0.05).5.In the expressing α+β-subunit BKCa,group without dexmedetomidine(control group),the To at different concentrations of dexmedetomidine(10-8-10-5 M)was gradually increased,NPo did not increase at the lower concentrations of dexmedetomidine(10-9-10-8 M)(P>0.05),while NPo gradually increased and had dose-dependent at higher dexmedetomidine concentrations(10-7-10-5 M)(P<0.05),but no significant differences in Tc,Amp(P>0.05).6.At the same calcium ion concentration,at the same dexmedetomidine concentration,compared with the expressing α-subunit BKCa(10-7 M),To and NPo was higher at the expressing α+β-subunit BKCa(P>0.05),no significant differences in Tc,Amp between expressing α-and α+β-subunit BKCa(P>0.05).Conclusion: 1.Under inside-out configuration,dexmedetomidine can activate BKCa of porcine coronary artery smooth muscle and exerts certain concentration dependence,which may be the mechanism of cardioprotection induced by dexmedetomidine.2.β subunit can elevate the voltage sensitivity of BKCa and calcium sensitivity.3.Under inside-out configuration,dexmedetomidine can activate expressing α-and α+β-subunit BKCa on HEK293 cells and demonstrate concentration-dependent.4.β subunit of BKCa can obviously enhance activation of BKCa on HEK293 cells induced by dexmedetomidine.