The Role Of Store-operated Calcium Entry And Sodium Calcium Exchanger In Carbachol-induced Tracheal Smooth Muscle Contraction

BackgroundRespiratory disease is a common disease and the main lesion in the trachea,bronchial smooth muscle.Calcium is a key factor in smooth muscle contraction.Calcium-store-operated calcium influx(store-operated Ca2+ entry,SOCE)is an important way to mediate Ca2+ influx.Orai1 and matrix-interacting protein molecule 1(STIM1)are two important components of store-operated calcium entry(SOCE).Orai1 is a four transmembrane protein which locating in the cell membrane.When depletion of calcium store mediated by thapsigargin(TG)induces STIM1 aggregation and interaction with Orai1 channel in plasma membrane,and then active Orai 1 channel and induce calcium influx.Sodium calcium exchanger(NCX)is a kind of cation transport protein which widely distributed in cytoplasmic membrane,mitochondrial membrane,endoplasmic reticulum and secretory vesicle membrane.NCX can regulate rapidly and accurately the intracellular Ca2+concentration,which influence the physiological activities such as intracellular signal transduction,cell growth and development.The relaxation and contraction of tracheal smooth muscle plays an important role in the regulation of airway diameter and respiratory function.Both SOCE and NCX contribute to the regulation of calcium concentration and systolic function in tracheal smooth muscle cells,but the detailed mechanism remains unclear.In this study,we used the mouse trachea as the research object,with NCX blockers KB-R7943,Orai1 blocker BTP2 and L-dependent voltage-dependent calcium channel blocker Nifedipine treatment,to explore its intrinsic mechanism.Objective To investigate the role of SOCE and NCX in the contraction of tracheal smooth muscle induced by carbachol.Methods1.Preparation of isolated tracheal rings Clean male C57 mice,aged 6-8 weeks and weighing 20-25 g,were sacrificed by overdose CO2,and the neck was fully exposed and the trachea was removed as soon as possible.(95%O2 and 5% CO2)in a Krebs solution.The trachea was cut quickly and accurately along the longitudinal axis into a tracheal ring of about 2 mm in length for using.2.In vitro tracheal tension test The prepared tracheal rings were carefully inserted into the double-layer hooks of the tension transducer,treated with 1 μmol/L carbachol and different types of blocking agents for10 min in the shrinking test,and finally 2.5 mmol/L Ca Cl2,the changes of tracheal ring tension during the experiment were recorded.3.Statistical analyses All data obtained in this study were expressed and analyzed using Sigma Plot 12.5software.Data were analyzed by independent sample t-test.P <0.05 was considered statistically significant.Results1.Blocking NCX inhibited significantly SOCE-induced tracheal smooth muscle contraction.2.Blocking voltage-dependent calcium channels had no significant effect on SOCE-induced tracheal smooth muscle contraction.3.Inhibition of Orai1 channel reduced significantly SOCE-induced tracheal smooth muscle contraction response,4.Blocking NCX inhibited significantly carbachol-induced contraction of tracheal smooth muscle.5.Blocking Orai1 significantly inhibited carbachol-induced contraction of tracheal smooth muscle.6.Blocking voltage-dependent calcium channels had no significant effect on carbachol-induced tracheal smooth muscle contraction.7.Blocking TRPC4 channel can significantly inhibit carbachol-induced tracheal smooth muscle contraction.Conclusion Both NCX and Orai1 were involved in carbachol-induced tracheal smooth muscle contraction,and Orai1 was involved in the initial phase of contraction indeuced by carbachol,which was involved in mediating carbachol-induced contraction.